Recombinant pinoresinol/lariciresinol reductase, recombinant dirigent protein, and methods of use

ABSTRACT

Dirigent proteins and pinoresinol/lariciresinol reductases have been isolated, together with cDNAs encoding dirigent proteins and pinoresinol/lariciresinol reductases. Accordingly, isolated DNA sequences are provided which code for the expression of dirigent proteins and pinoresinol/lariciresinol reductases. In other aspects, replicable recombinant cloning vehicles are provided which code for dirigent proteins or pinoresinol/lariciresinol reductases or for a base sequence sufficiently complementary to at least a portion of dirigent protein or pinoresinol/lariciresinol reductase DNA or RNA to enable hybridization therewith. In yet other aspects, modified host cells are provided that have been transformed, transfected, infected and/or injected with a recombinant cloning vehicle and/or DNA sequence encoding dirigent protein or pinoresinol/lariciresinol reductase. Thus, systems and methods are provided for the recombinant expression of dirigent proteins and/or pinoresinol/lariciresinol reductases.

RELATED APPLICATIONS

The present application is a continuation-in-part of copending U.S. patent application Ser. No. 09/307,653, filed May 7, 1999, which claims the benefit of priority of international application PCT/US97/20391, filed Nov. 7, 1997, which claims the benefit of priority of U.S. provisional application serial No. 60/030,522, filed Nov. 8, 1996, and of U.S. provisional application serial No. 60/054,380, filed Jul. 31, 1997, the benefit of which is hereby claimed under 35 U.S.C., sections 119 and 120.

GOVERNMENT RIGHTS

This invention was funded in part by grant number DE-FG03-97ER20259 from the United States Department of Energy, by grant number MCB09631980 from the National Science Foundation, by grant number NAG100164 from the National Aeronautics and Space Administration, and by grant number 96-35103-3358 from the United States Department of Agriculture. The government has certain rights in this invention.

FIELD OF THE INVENTION

The present invention relates to isolated dirigent proteins from Forsythia intermedia, Tsuga heterophylla, Thuja plicata, Eucommia ulmoides, and Schisandra chinensis and to isolated pinoresinol/lariciresinol reductases from Forsythia intermedia, Tsuga heterophylla, Linum usitatissimum, and Schisandra chinensis. The present invention also relates to nucleic acid sequences which code for dirigent proteins from Forsythia intermedia, Tsuga heterophylla, Thuja plicata, Eucommia ulmoides, and Schisandra chinensis and to nucleic acid sequences which code for pinoresinol/lariciresinol reductases from Forsythia intermedia, Tsuga heterophylla, Thuja plicata, Eucommia ulmoides, and Schisandra chinensis. The present invention also relates to vectors containing the sequences, host cells containing the sequences and methods of producing recombinant pinoresinol/lariciresinol reductases, recombinant dirigent proteins and their mutants.

BACKGROUND OF THE INVENTION

Lignans are a large, structurally diverse, class of vascular plant metabolites having a wide range of physiological functions and pharmacologically important properties (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990); Lewis et al., in Chemistry of the Amazon, Biodiversity Natural Products, and Environmental Issues, 588, (P. R. Seidl, O. R. Gottlieb and M. A. C. Kaplan) 135-167, ACS Symposium Series, Washington D.C. (1995)). Because of their pronounced antibiotic properties (Markkanen, T. et al., Drugs Exptl. Clin. Res. 7:711-718 (1981)), antioxidant properties (Fauré, M. et al., Phytochemistry 29:3773-3775 (1990); Osawa, T. et al., Agric. Biol. Chem. 49:3351-3352 (1985)) and antifeedant properties (Harmatha, J., and Nawrot, J., Biochem. Syst. Ecol. 12:95-98 (1984)), a major role of lignans in vascular plants is to help confer resistance against various opportunistic biological pathogens and predators. Lignans have also been proposed as cytokinins (Binns, A. N. et al., Proc. Natl. Acad. Sci. USA 84:980-984 (1987)) and as intermediates in lignification (Rahman, M. M. A. et al., Phytochemistry 29:1861-1866 (1990)), suggesting a critical role in plant growth and development. It is widely held that elaboration of biochemical pathways to lignins/lignans and related substances from phenylalanine (tyrosine) was essential for the successful transition of aquatic plants to their vascular dry-land counterparts (Lewis, N. G., and Davin, L. B., in Isoprenoids and Other Natural Products. Evolution and Function, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, DC (1994)), some four hundred and eighty million years ago (Graham, L. E., Origin of Land Plants, John Wiley & Sons, Inc., New York, N.Y. (1993)).

Based on existing chemotaxonomic data, lignans are present in “primitive” plants, such as the fern Blechnum orientate (Wada, H. et al., Chem. Pharm. Bull. 40:2099-2101 (1992)) and the hornworts, e.g., Dendroceros japonicus and Megaceros flagellaris (Takeda, R. et al., in Bryophytes. Their Chemistry and Chemical Taxonomy, Vol. 29 (Zinsmeister, H. D. and Mues, R. eds) pp. 201-207, Oxford University Press: New York, N.Y. (1990); Takeda, R. et al., Tetrahedron Lett. 31:4159-4162 (1990)), with the latter recently being classified as originating in the Silurian period (Graham, L. E., J. Plant Res. 109: 241-252 (1996)). Interestingly, evolution of both gymnosperms and angiosperms was accompanied by major changes in the structural complexity and oxidative modifications of the lignans (Lewis, N. G., and Davin, L. B., in Isoprenoids and Other Natural Products, Evolution and Function, 562 (W. D. Nes, ed) 202-246, ACS Symposium Series: Washington, DC (1994); Gottlieb, O. R., and Yoshida, M., in Natural Products of Woody Plants. Chemicals Extraneous to the Lignocellulosic Cell Wall (Rowe, J. W. and Kirk, C. H. eds) pp. 439-511, Springer Verlag: Berlin (1989)). Indeed, in some species, such as Western Red Cedar (Thuja plicata), lignans can contribute extensively to heartwood formation/generation by enhancing the resulting heartwood color, quality, fragrance and durability.

In addition to their functions in plants, lignans also have important pharmacological roles. For example, podophyllotoxin, as its etoposide and teniposide derivatives, is an example of a plant compound that has been successfully employed as an anticancer agent (Ayres, D. C., and Loike, J. D. in Chemistry and Pharmacology of Natural Products. Lignans. Chemical, Biological and Clinical Properties, Cambridge University Press, Cambridge, England (1990)). Antiviral properties have also been reported for selected lignans. For example, (−)-arctigenin (Schröder, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)), (−)-trachelogenin (Schröder, H. C. et al., Z. Naturforsch. 45c, 1215-1221 (1990)) and nordihydroguaiaretic acid (Gnabre, J. N. et al., Proc. Natl. Acad. Sci. USA 92:11239-11243 (1995)) are each effective against HIV due to their pronounced reverse transcriptase inhibitory activities. Some lignans, e.g., matairesinol (Nikaido, T. et al., Chem. Pharm. Bull. 29:3586-3592 (1981)), inhibit cAMP-phosphodiesterase, whereas others enhance cardiovascular activity, e.g., syringaresinol β-D-glucoside (Nishibe, S. et al., Chem. Pharm. Bull. 38:1763-1765 (1990)). There is also a high correlation between the presence, in the diet, of the “mammalian” lignans or “phytoestrogens”, enterolactone and enterodiol, formed following digestion of high fiber diets, and reduced incidence rates of breast and prostate cancers (so-called chemoprevention) (Axelson, M., and Setchell, K. D. R., FEBS Lett. 123:337-342 (1981); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 41:3-8 (1992); Adlercreutz et al., J. Steroid Biochem. Molec. Biol. 52:97-103 (1995)). The “mammalian lignans,” in turn, are considered to be derived from lignans such as matairesinol and secoisolariciresinol (Boriello et al., J. Applied Bacteriol., 58:37-43 (1985)).

The biosynthetic pathways to the lignans are only now being defined, although there are no prior art reports of the isolation of enzymes or genes involved in the lignan biosynthetic pathway. Based on radiolabeling experiments with crude enzyme extracts from Forsythia intermedia, it was first established that entry into the 8,8′-linked lignans, which represent the most prevalent dilignol linkage known (Davin, L. B., and Lewis, N. G., in Rec. Adv. Phytochemistry, Vol. 26 (Stafford, H. A., and Ibrahim, R. K., eds), pp. 325-375, Plenum Press, New York, N.Y. (1992)), occurs via stereoselective coupling of two achiral coniferyl alcohol molecules, in the form of oxygenated free radicals, to afford the furofuran lignan (+)-pinoresinol (Davin, L. B., Bedgar, D. L., Katayama, T., and Lewis, N. G., Phytochemistry 31:3869-3874 (1992); Paré, P. W. et al., Tetrahedron Lett. 35:4731-4734 (1994)) (FIG. 1).

Bimolecular phenoxy radical coupling reactions, such as the stereoselective coupling of two achiral coniferyl alcohol molecules to afford the furofuran lignan (+)-pinoresinol, are involved in numerous biological processes. These are presumed to include lignin formation in vascular plants (M. Nose et al., Phytochemistry 39:71 (1995)), lignan formation in vascular plants (N. G. Lewis and L. B. Davin, ACS Symp. Ser. 562:202 (1994); P. W. Paré et al., Tetrahedron Lett. 35:4731 (1994)), suberin formation in vascular plants (M. A. Bernards et al., J. Biol. Chem. 270:7382 (1995)), fruiting body development in fungi (J. D. Bu'Lock et al., J. Chem. Soc. 2085 (1962)), insect cuticle melanization and sclerotization (M. Miessner et al., Helv. Chim. Acta 74:1205 (1991); V. J. Marmaras et al., Arch. Insect Biochem. Physiol. 31:119 (1996)), the formation of aphid pigments (D. W. Cameron and Lord Todd, in Organic Substances of Natural Origin. Oxidative Coupling of Phenols, W. I. Taylor and A. R. Battersby, Eds. (Dekker, New York, 1967), Vol. 1, p.203), and the formation of algal cell wall polymers (M. A. Ragan, Phytochemistry 23:2029 (1984)).

In contrast to the marked regiochemical and/or stereochemical specificities observed in the biosynthesis of the foregoing lignin and lignan substances in vivo, all previously described chemical (J. Iqbal et al., Chem. Rev. 94:519 (1994)) and enzymatic (K. Freudenberg, Science 148:595 (1965)) bimolecular phenoxy radical coupling reactions in vitro have lacked strict regio- and stereospecific control. That is, if chiral centers are introduced during coupling in vitro, the products are racemic, and different regiochemistries can result if more than one potential coupling site is present. Thus, the ability to generate a particular enantiomeric form or a specific coupling product in vitro is not under explicit control. Consequently, it is inferred that a mechanism exists in vivo to control the regiochemistry and stereochemistry of bimolecular phenoxy radical coupling reactions leading to the formation of, for example, lignans.

In Forsythia intermedia, and presumably other species, (+)-pinoresinol, the product of the stereospecific coupling of two E-coniferyl alcohol molecules, undergoes sequential reduction to generate (+)-lariciresinol and then (−)-secoisolariciresinol (Katayama, T. et al., Phytochemistry 32:581-591 (1993); Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)) (FIG. 1). While it has hitherto been unclear whether more than one reductase is required to catalyze the sequential steps, the reductions proceed via abstraction of the pro-R hydride of NADPH, resulting in an “inversion” of configuration at both the C-7 and C-7′ positions of the products, (+)-lariciresinol and (−)-secoisolariciresinol (Chu, A., et al., J. Biol. Chem. 268:27026-27033 (1993)). (−)-Matairesinol is subsequently formed via dehydrogenation of (−)-secoisolariciresinol, further metabolism of which presumably affords lignans such as the antiviral (−)-trachelogenin in Ipomoea cairica and (−)-podophyllotoxin in Podophyllum peltatum.

Thus, the stereospecific formation of (+)-pinoresinol and the subsequent reductive steps giving (+)-lariciresinol and (−)-secoisolariciresinol are pivotal points in lignan metabolism, since they represent entry into the furano, dibenzylbutane, dibenzylbutyrolactone and aryltetrahydronaphthalene lignan subclasses. Additionally, it should be noted that while lignans are normally optically active, the particular enantiomer present may differ between plant species. For example, (−)-pinoresinol occurs in Xanthoxylum ailanthoides (Ishii et al., Yakugaku Zasshi, 103:279-292 (1983)), and (−)-lariciresinol is present in Daphne tangutica (Lin-Gen, et al., Planta Medica, 45:172-176 (1982)). The optical activity of a particular lignan may have important ramifications regarding biological activity. For example, (−)-trachelogenin inhibits the in vitro replication of HIV-1, whereas its (+)-enantiomer is much less effective (Schroder et al., Naturforsch. 45c:1215-1221(1990)).

SUMMARY OF THE INVENTION

In accordance with the foregoing, in one aspect of the invention it has now been discovered that a 78-kD dirigent protein is involved in conferring stereospecificity in 8,8′-linked lignan formation. This protein has no detectable catalytically active oxidative center and apparently serves only to bind and orient coniferyl alcohol-derived free radicals, which then undergo stereoselective coupling to form (+)-pinoresinol. The formation of free-radicals, in the first instance, requires the oxidative capacity of either a nonspecific oxidase or even a non-enzymatic electron oxidant. In another aspect of the invention, it has been discovered that a single enzyme, designated pinoresinol/lariciresinol reductase, catalyzes the conversion of pinoresinol to lariciresinol and then to secoisolariciresinol. Thus, one aspect of the invention relates to isolated dirigent proteins and to isolated pinoresinol/lariciresinol reductases, such as, for example, those from Forsythia intermedia, Thuja plicata, Tsuga heterophylla, Eucommia ulmoides, Schisandra chinensis, and Linum usitatissimum.

In other aspects of the invention, cDNAs encoding dirigent protein from several plant species have been isolated and sequenced, and the corresponding amino acid sequences have been deduced. Also, cDNAs encoding pinoresinol/lariciresinol reductase from several plant species have been isolated and sequenced, and the corresponding amino acid sequences have been deduced.

Thus, the present invention relates to isolated proteins and to isolated DNA sequences which code for the expression of dirigent protein or pinoresinol/lariciresinol reductase. In other aspects, the present invention is directed to replicable vectors comprising a nucleic acid sequence which codes for a pinoresinol/lariciresinol reductase or for a dirigent protein. The present invention is also directed to a base sequence sufficiently complementary to at least a portion of a pinoresinol/lariciresinol reductase DNA or RNA, or to at least a portion of a dirigent protein DNA or RNA, to enable hybridization therewith. The aforesaid complementary base sequences include, but are not limited to: antisense pinoresinol/lariciresinol reductase RNA; antisense dirigent protein RNA; fragments of DNA that are complementary to a pinoresinol/lariciresinol reductase DNA, or to a dirigent protein DNA, and which are therefore useful as polymerase chain reaction primers, or as probes for pinoresinol/lariciresinol reductase genes, dirigent protein genes, or related genes.

In yet other aspects of the invention, modified host cells are provided that have been transformed, transfected, infected and/or injected with a replicable vector and/or DNA sequence of the invention. Thus, the present invention provides for the recombinant expression of pinoresinol/lariciresinol reductases and dirigent proteins in plants, animals, microbes and in cell cultures. The inventive concepts described herein may be used to facilitate the production, isolation and purification of significant quantities of recombinant pinoresinol/lariciresinol reductase or dirigent protein, or of their enzyme products, in plants, animals, microbes or cell cultures.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing aspects and many of the attendant advantages of this invention will become more readily appreciated as the same becomes better understood by reference to the following detailed description, when taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the stereospecific conversion of E-coniferyl alcohol to (+)-pinoresinol in Forsythia intermedia. The stereoselectivity of this reaction is controlled by dirigent protein. (+)-Pinoresinol is then sequentially converted to (+)-lariciresinol and (−)-secoisolariciresinol by (+)-pinoresinol/(+)-lariciresinol reductase. (+)-pinoresinol, (+)-lariciresinol and (−)-secoisolariciresinol are the precursors of the furofuran, furano and dibenzylbutane families of lignans, respectively.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

As used herein, the terms “amino acid” and “amino acids” refer to all naturally occurring L-α-amino acids or their residues. The amino acids are identified by either the single-letter or three-letter designations:

Asp D aspartic acid Ile I isoleucine Thr T threonine Leu L leucine Ser S serine Tyr Y tyrosine Glu F glutamic acid Phe F phenylalanine Pro P proline His H histidine Gly G glycine Lys K lysine Ala A alanine Arg R arginine Cys C cysteine Trp W tryptophan Val V valine Gln Q glutamine Met M methionine Asn N asparagine

As used herein, the term “nucleotide” means a monomeric unit of DNA or RNA containing a sugar moiety (pentose), a phosphate and a nitrogenous heterocyclic base. The base is linked to the sugar moiety via the glycosidic carbon (1′ carbon of pentose) and that combination of base and sugar is called a nucleoside. The base characterizes the nucleotide with the four bases of DNA being adenine (“A”), guanine (“G”), cytosine (“C”) and thymine (“T”). Inosine (“I”) is a synthetic base that can be used to substitute for any of the four, naturally-occurring bases (A, C, G or T). The four RNA bases are A,G,C and uracil (“U”). The nucleotide sequences described herein comprise a linear array of nucleotides connected by phosphodiester bonds between the 3′ and 5′ carbons of adjacent pentoses.

The term “percent identity” (% I) means the percentage of amino acids or nucleotides that occupy the same relative position when two amino acid sequences, or two nucleic acid sequences, are aligned side by side.

The term “percent similarity” (% S) is a statistical measure of the degree of relatedness of two compared protein sequences. The percent similarity is calculated by a computer program that assigns a numerical value to each compared pair of amino acids based on chemical similarity (e.g., whether the compared amino acids are acidic, basic, hydrophobic, aromatic, etc.) and/or evolutionary distance as measured by the minimum number of base pair changes that would be required to convert a codon encoding one member of a pair of compared amino acids to a codon encoding the other member of the pair. Calculations are made after a best fit alignment of the two sequences has been made empirically by iterative comparison of all possible alignments. (Henikoff, S. and Henikoff, J. G., Proc. Nat'l Acad Sci USA 89:10915-10919 (1992)).

“Oligonucleotide” refers to short length single or double stranded sequences of deoxyribonucleotides linked via phosphodiester bonds. The oligonucleotides are chemically synthesized by known methods and purified, for example, on polyacrylamide gels.

The term “pinoresinol/lariciresinol reductase” is used herein to mean an enzyme capable of catalyzing two reduction reactions: the reduction of pinoresinol to lariciresinol, and the reduction of lariciresinol to secoisolariciresinol. The products of these reactions, lariciresinol and secoisolariciresinol, can be either the (+)- or (−)-enantiomers.

The term “dirigent protein” is used herein to mean a protein capable of guiding a bimolecular phenoxy radical coupling reaction thereby determining the stereochemistry and regiochemistry of the product of the reaction and/or its polymeric derivatives.

The term “stringent wash conditions” refers to the conditions used to wash a nucleic acid blot, such as a Southern blot. The following are representative hybridization and stringent wash conditions useful for identifying (by Southern blotting) nucleic acid molecules of the invention that are capable of hybridizing to a nucleic acid molecule selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 77, 86, 93, 98 and 105, or to the antisense complement of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 77, 86, 93, 98 and 105: hybridization in 6×SSC, 5×Denhardt's, 0.5% SDS at 55-58° C. for 12 hours, followed by washing in 2×SSC, 0.5% SDS at 55-58° C. for 30 minutes. An optional further wash can be conducted in 1×SSC, 0.5% SDS at 55-58° C. for 30 minutes, followed by an additional, optional wash in 0.5×SSC, 0.5% SDS at 55-58° C. for 30 minutes.

The following are representative hybridization and stringent wash conditions useful for identifying (by Southern blotting) nucleic acid molecules of the invention that are capable of hybridizing to a nucleic acid molecule selected from the group consisting of SEQ ID NOS:47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71, 107 and 117, or to the antisense complement of a nucleic acid molecule selected from the group consisting of SEQ ID NOS:47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71, 107 and 117: hybridization in 6×SSC, 5×Denhardt's, 0.5% SDS at 57-58° C. for 12 hours, followed by one wash in 4×SSC, 0.5% SDS at room temperature (typically 20° C. to 30° C.) for 5 minutes, followed by one wash in 2×SSC, 0.5% SDS at 57-58° C. for 20 minutes. An optional further wash can be conducted in 1×SSC, 0.5% SDS at 57-58° C. for 30 minutes, followed by an additional, optional wash in 0.5×SSC, 0.5% SDS at 57-58° C. for 30 minutes.

The terms “alteration”, “amino acid sequence alteration”, “variant” and “amino acid sequence variant” refer to dirigent protein or pinoresinol/lariciresinol reductase molecules with some differences in their amino acid sequences as compared to the corresponding native dirigent protein or pinoresinol/lariciresinol reductase. Ordinarily, the variants will possess at least about 70% homology with the corresponding, native dirigent protein or pinoresinol/lariciresinol reductase, and preferably they will be at least about 80% homologous with the corresponding, native dirigent protein or pinoresinol/lariciresinol reductase. The amino acid sequence variants of dirigent protein or pinoresinol/lariciresinol reductase falling within this invention possess substitutions, deletions, and/or insertions at certain positions. Sequence variants of dirigent protein or pinoresinol/lariciresinol reductase may be used to attain desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution.

Substitutional dirigent protein variants or pinoresinol/lariciresinol reductase variants are those that have at least one amino acid residue in the corresponding native dirigent protein sequence or pinoresinol/lariciresinol reductase sequence removed and a different amino acid inserted in its place at the same position. The substitutions may be single, where only one amino acid in the molecule has been substituted, or they may be multiple, where two or more amino acids have been substituted in the same molecule. Substantial changes in the activity of the dirigent protein or pinoresinol/lariciresinol reductase molecule may be obtained by substituting an amino acid with a side chain that is significantly different in charge and/or structure from that of the native amino acid. This type of substitution would be expected to affect the structure of the polypeptide backbone and/or the charge or hydrophobicity of the molecule in the area of the substitution.

Moderate changes in the activity of the dirigent protein or pinoresinol/lariciresinol reductase molecule would be expected by substituting an amino acid with a side chain that is similar in charge and/or structure to that of the native molecule. This type of substitution, referred to as a conservative substitution, would not be expected to substantially alter either the structure of the polypeptide backbone or the charge or hydrophobicity of the molecule in the area of the substitution.

Insertional dirigent protein variants or pinoresinol/lariciresinol reductase variants are those with one or more amino acids inserted immediately adjacent to an amino acid at a particular position in the native dirigent protein or pinoresinol/lariciresinol reductase molecule. Immediately adjacent to an amino acid means connected to either the α-carboxy or α-amino functional group of the amino acid. The insertion may be one or more amino acids. Ordinarily, the insertion will consist of one or two conservative amino acids. Amino acids similar in charge and/or structure to the amino acids adjacent to the site of insertion are defined as conservative. Alternatively, this invention includes insertion of an amino acid with a charge and/or structure that is substantially different from the amino acids adjacent to the site of insertion.

Deletional variants are those where one or more amino acids in the native dirigent protein or pinoresinol/lariciresinol reductase molecule have been removed. Ordinarily, deletional variants will have one or two amino acids deleted in a particular region of the dirigent protein or pinoresinol/lariciresinol reductase molecule.

The term “antisense” or “antisense RNA” or “antisense nucleic acid” is used herein to mean a nucleic acid molecule that is complementary to all or part of a messenger RNA molecule. Antisense nucleic acid molecules are typically used to inhibit the expression, in vivo, of complementary, expressed messenger RNA molecules.

The terms “biological activity”, “biologically active”, “activity” and “active” when used with reference to a pinoresinol/lariciresinol reductase molecule refer to the ability of the pinoresinol/lariciresinol reductase molecule to reduce pinoresinol and lariciresinol to yield lariciresinol and secoisolariciresinol, respectively, as measured in an enzyme activity assay, such as the assay described in Example 8 below.

The terms “biological activity”, “biologically active”, “activity” and “active” when used with reference to a dirigent protein refer to the ability of the dirigent protein to guide a bimolecular phenoxy radical coupling reaction thereby determining the stereochemistry and regiochemistry of the product of the reaction and of its polymeric derivatives.

Amino acid sequence variants of dirigent protein or pinoresinol/lariciresinol reductase may have desirable altered biological activity including, for example, altered reaction kinetics, substrate utilization, product distribution or other characteristics such as regiochemistry and stereochemistry.

The terms “DNA sequence encoding”, “DNA encoding” and “nucleic acid encoding” refer to the order or sequence of deoxyribonucleotides along a strand of deoxyribonucleic acid. The order of these deoxyribonucleotides determines the order of amino acids along the translated polypeptide chain. The DNA sequence thus codes for the amino acid sequence.

The term “replicable vector” refers to a piece of DNA, usually double-stranded, which may have inserted into it a piece of foreign DNA. Foreign DNA is defined as heterologous DNA, which is DNA not naturally found in the host. The vector is used to transport the foreign or heterologous DNA into a suitable host cell. Once in the host cell, the vector can replicate independently of or coincidentally with the host chromosomal DNA, and several copies of the vector and its inserted (foreign) DNA may be generated. The term “replicable vector” includes replicable expression vectors that contain the necessary elements that permit translating the foreign DNA into a polypeptide. Many molecules of the polypeptide encoded by the foreign DNA can thus be rapidly synthesized. Replicable vectors can also include insert DNA that is normally found in the host.

The terms “transformed host cell,” “transformed” and “transformation” refer to the introduction of DNA into a cell. The cell is termed a “host cell”, and it may be a prokaryotic or a eukaryotic cell. Typical prokaryotic host cells include various strains of E. coli. Typical eukaryotic host cells are plant cells, such as maize cells, yeast cells, insect cells or animal cells. The introduced DNA is usually in the form of a vector containing an inserted piece of DNA. The introduced DNA sequence may be from the same species as the host cell or from a different species from the host cell, or it may be a hybrid DNA sequence, containing some foreign DNA and some DNA derived from the host species.

In accordance with the present invention, cDNAs encoding dirigent protein and pinoresinol/lariciresinol reductase from several plant species, including Forsythia intermedia, Thuja plicata Tsuga heterophylla, Eucommia ulmoides, Schisandra chinensis, and Linum usitatissimum were isolated, sequenced and expressed in the following manner.

With respect to the cDNAs encoding dirigent protein from Forsythia intermedia, an empirically-determined purification protocol was developed to isolate the Forsythia dirigent protein. This procedure yielded at least six isoforms of the dirigent protein. Amino acid sequencing of the amino terminus of each of these isoforms revealed that the sequence of each isoform was identical. Sequencing of the N-terminus of a mixture of these isoforms yielded a 28 amino acid sequence (SEQ ID NO:1). Tryptic digestion of a mixture of these isoforms yielded six peptide fragments which were purified in sufficient quantity to permit sequencing SEQ ID NOS:2-7.

A primer designated PSINT1 (SEQ ID NO:8) was synthesized based on the sequence of amino acids 9 to 15 of the N-terminal peptide (SEQ ID NO:1). A primer designated PSI1R (SEQ ID NO:9) was synthesized based on the sequence of amino acids 3 to 9 of the internal peptide sequence set forth in (SEQ ID NO:2). A primer designated PSI2R (SEQ ID NO:10) was synthesized based on the sequence of amino acids 13 to 20 of the internal peptide sequence set forth in (SEQ ID NO:2). A primer designated PSI7R (SEQ ID NO:11) was synthesized based on the sequence of amino acids 6 to 12 of the internal peptide sequence set forth in (SEQ ID NO:3).

Forsythia total RNA was isolated by means of a protocol adapted from a method specifically designed for woody tissues which contain a large concentration of polyphenols. Poly A+RNA was isolated and a cDNA library constructed using standard means. A PCR reaction utilizing primers PSINT1 (SEQ ID NO:8) and one of PSI7R, (SEQ ID NO:11) PSI2R (SEQ ID NO:10) or PSI1R (SEQ ID NO:9), together with an aliquot of Forsythia cDNA as substrate, each yielded a single cDNA band of ˜370 bp, ˜155 bp and ˜125 bp, respectively. The ˜370 bp product of the PSINT1 (SEQ ID NO:8)-PSI7R (SEQ ID NO:11) reaction was amplified by PCR and utilized as a probe to screen approximately 600,000 PFU of a Forsythia intermedia cDNA library. Two distinct cDNAs were identified, called pPSDFi1 (SEQ ID NO:12) and pPSDFi2 (SEQ ID NO:14). The cDNA insert encoding dirigent protein was excised from plasmid pPSDFi1 and cloned into the baculovirus transfer vector pBlueBac4. The resulting construct was used to transform Spodoptera frugiperda from which functional dirigent protein was purified.

In another aspect, Forsythia cDNAs were used as probes to isolate two dirigent protein clones from Tsuga heterophylla (SEQ ID NOS:16, 18), and eight dirigent protein cDNA clones from Thuja plicata (SEQ ID NOS:20, 22, 24, 26, 28, 30, 32, 34).

With respect to the cDNAs encoding (+)-pinoresinol/(+)-lariciresinol reductase from Forsythia intermedia, an empirically-determined purification protocol, consisting of eight chromatographic steps, was developed to isolate the Forsythia (+)-pinoresinol/(+)-lariciresinol reductase protein. This procedure yielded two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase which were both capable of catalyzing the reduction of (+)-pinoresinol and (+)-lariciresinol. Sequencing of the N-terminus of each of these isoforms yielded an identical 30 amino acid sequence (SEQ ID NO:36). Tryptic digestion of a mixture of both of these isoforms yielded four peptide fragments which were purified in sufficient quantity to permit sequencing (SEQ ID Nos:37-40). Additionally, cyanogen bromide cleavage of a mixture of both of these isoforms yielded three peptide fragments which were purified in sufficient quantity to permit sequencing (SEQ ID Nos:41-43).

A primer designated PLRN5 (SEQ ID NO:44) was synthesized based on the sequence of amino acids 7 to 13 of the N-terminal peptide (SEQ ID NO:36). A primer designated PLR14R (SEQ ID NO:45) was synthesized based on the sequence of amino acids 2 to 8 of the internal peptide sequence set forth in SEQ ID NO:37. A primer designated PLR15R (SEQ ID NO:46) was synthesized based on the sequence of amino acids 9 to 15 of the internal peptide sequence set forth in SEQ ID NO:37. The sequence of amino acids 9 to 15 of the internal peptide sequence set forth in SEQ ID NO:37, upon which the sequence of primer PLR15R (SEQ ID NO:46) was based, also corresponded to the sequence of amino acids 4 to 10 of the cyanogen bromide-generated, internal fragment set forth in SEQ ID NO:41.

Forsythia total RNA was isolated by means of a protocol adapted from a method specifically designed for woody tissues which contain a large concentration of polyphenols. Poly A+RNA was isolated and a cDNA library constructed using standard means. A PCR reaction utilizing primers PLRN5 (SEQ ID NO:44) and either PLR14R (SEQ ID NO:45) or PLR15R (SEQ ID NO:46), together with an aliquot of Forsythia cDNA as substrate, yielded two, amplified bands of 380 bp and 400 bp. One 400 bp cDNA insert was utilized as a probe with which to screen the Forsythia cDNA library. The 400 bp probe corresponded to bases 22 to 423 of SEQ ID NO:47. Six cDNA clones were isolated and sequenced (SEQ ID Nos:47, 49, 51, 53, 55, 57). The clones shared a common coding region, many had a different 5′-untranslated region and the 3′-untranslated region of each terminated at a different point. One of these cDNAs (SEQ ID NO:47), expressed as a β-galactosidase fusion protein in E. coli, catalyzed the same enantiomer-specific reactions as the native plant protein.

In another aspect, (+)-pinoresinol/(+)-lariciresinol reductase and (−)-pinoresinol/(−)-lariciresinol reductase from Thuja plicata were isolated by synthesizing Thuja plicata cDNA which was utilized as a template in a PCR reaction in which the primers were a 3′ linker-primer (SEQ ID NO:59) and a 5′ primer, designated CR6-NT, (SEQ ID NO:60). At least two bands of the expected length (1.2 kb) were generated and cloned into a plasmid vector. One clone, designated plr-Tp1, (SEQ ID NO:61) was completely sequenced and expressed as a β-galactosidase fusion protein in E. coli. plr-Tp1 (SEQ ID NO:61) encodes a (−)-pinoresinol/(−)-lariciresinol reductase.

The cDNA insert of clone plr-Tp1 (SEQ ID NO:61) was used to screen the T. plicata cDNA library and identified an additional, unique clone, designated plr-Tp2, (SEQ ID NO:63). plr-Tp2 (SEQ ID NO:63) has high homology to plr-Tp1 (SEQ ID NO:61) but encodes a (+)-pinoresinol/(+)-lariciresinol reductase. The cDNA insert of clone plr-Tp1 (SEQ ID NO:61) was used to screen the T. plicata cDNA library and identify an additional two pinoresinol/lariciresinol reductase cDNAs (SEQ ID NOS:65, 67).

Two cDNAs encoding pinoresinol/lariciresinol reductases from Tsuga heterophylla (SEQ ID Nos:69, 71) were isolated by screening a Tsuga heterophylla cDNA library with the plr-Tp1 cDNA insert (SEQ ID NO:61). Additional pinoresinol/lariciresinol reductase cDNAs and dirigent protein cDNAs were isolated as described in Examples 17-22 herein.

The isolation of cDNAs encoding dirigent proteins, (+)-pinoresinol/(+)-lariciresinol reductase and (−)-pinoresinol/(−)-lariciresinol reductase permits the development of an efficient expression system for these functional enzymes; provides useful tools for examining the developmental regulation of lignan biosynthesis and permits the isolation of other dirigent proteins and pinoresinol/lariciresinol reductases. The isolation of the dirigent protein and pinoresinol/lariciresinol reductase cDNAs also permits the transformation of a wide range of organisms in order to enhance or modify lignan biosynthesis.

The proteins and nucleic acids of the present invention can be utilized to predetermine the stereochemistry, regiochemistry, or both, of the products of bimolecular phenoxy coupling reactions, such as the furofuran, furano and dibenzylbutane lignans. By way of non-limiting examples, the proteins and nucleic acids of the present invention can be utilized to: elevate or otherwise alter the levels of health-protecting lignans, such as podophyllotoxin, in plant species, including but not limited to vegetables, grains and fruits, and to food items incorporating material derived from such genetically altered plants; genetically alter plant species to provide an abundant, natural supply of lignans useful for a variety of purposes, for example as neutriceuticals and dietary supplements; to genetically alter living organisms to produce an abundant supply of optically pure lignans having desirable biological properties, for example (−)-arctigenin which possesses antiviral properties. In particular, characterization of the dirigent protein binding site and mechanism of action permits the development of synthetic proteins consisting of an array of dirigent protein binding sites which serve as templates for stereochemically-controlled polymeric assembly.

N-terminal transport sequences well known in the art (see, e.g., von Heijne, G. et al., Eur. J. Biochem 180:535-545 (1989); Stryer, Biochemistry W. H. Freeman and Company, New York, N.Y., p. 769 (1988)) may be employed to direct the dirigent protein or pinoresinol/lariciresinol reductase to a variety of cellular or extracellular locations.

Sequence variants of wild-type dirigent protein clones and pinoresinol/lariciresinol clones that can be produced by deletions, substitutions, mutations and/or insertions are intended to be within the scope of the invention except insofar as limited by the prior art. Dirigent protein or pinoresinol/lariciresinol reductase amino acid sequence variants may be constructed by mutating the DNA sequence that encodes wild-type dirigent protein or wild-type pinoresinol/lariciresinol reductase, such as by using techniques commonly referred to as site-directed mutagenesis. Various polymerase chain reaction (PCR) methods now well known in the field, such as a two primer system like the Transformer Site-Directed Mutagenesis kit from Clontech, may be employed for this purpose.

Following denaturation of the target plasmid in this system, two primers are simultaneously annealed to the plasmid; one of these primers contains the desired site-directed mutation, the other contains a mutation at another point in the plasmid resulting in elimination of a restriction site. Second strand synthesis is then carried out, tightly linking these two mutations, and the resulting plasmids are transformed into a mutS strain of E. coli. Plasmid DNA is isolated from the transformed bacteria, restricted with the relevant restriction enzyme (thereby linearizing the unmutated plasmids), and then retransformed into E. coli. This system allows for generation of mutations directly in an expression plasmid, without the necessity of subcloning or generation of single-stranded phagemids. The tight linkage of the two mutations and the subsequent linearization of unmutated plasmids results in high mutation efficiency and allows minimal screening. Following synthesis of the initial restriction site primer, this method requires the use of only one new primer type per mutation site. Rather than prepare each positional mutant separately, a set of “designed degenerate” oligonucleotide primers can be synthesized in order to introduce all of the desired mutations at a given site simultaneously. Transformants can be screened by sequencing the plasmid DNA through the mutagenized region to identify and sort mutant clones. Each mutant DNA can then be restricted and analyzed by electrophoresis on Mutation Detection Enhancement gel (J. T. Baker) to confirm that no other alterations in the sequence have occurred (by band shift comparison to the unmutagenized control).

The verified mutant duplexes can be cloned into a replicable expression vector, if not already cloned into a vector of this type, and the resulting expression construct used to transform E. coli, such as strain E. coli BL21(DE3)pLysS, for high level production of the mutant protein, and subsequent purification thereof. The method of FAB-MS mapping can be employed to rapidly check the fidelity of mutant expression. This technique provides for sequencing segments throughout the whole protein and provides the necessary confidence in the sequence assignment. In a mapping experiment of this type, protein is digested with a protease (the choice will depend on the specific region to be modified since this segment is of prime interest and the remaining map should be identical to the map of unmutagenized protein). The set of cleavage fragments is fractionated by microbore HPLC (reversed phase or ion exchange, again depending on the specific region to be modified) to provide several peptides in each fraction, and the molecular weights of the peptides are determined by FAB-MS. The masses are then compared to the molecular weights of peptides expected from the digestion of the predicted sequence, and the correctness of the sequence quickly ascertained. Since this mutagenesis approach to protein modification is directed, sequencing of the altered peptide should not be necessary if the MS agrees with prediction. If necessary to verify a changed residue, CAD-tandem MS/MS can be employed to sequence the peptides of the mixture in question, or the target peptide purified for subtractive Edman degradation or carboxypeptidase Y digestion depending on the location of the modification.

In the design of a particular site directed mutant, it is generally desirable to first make a non-conservative substitution (e.g., Ala for Cys, His or Glu) and determine if activity is greatly impaired as a consequence. The properties of the mutagenized protein are then examined with particular attention to the kinetic parameters of K_(m) and k_(cat) as sensitive indicators of altered function, from which changes in binding and/or catalysis per se may be deduced by comparison to the native enzyme. If the residue is by this means demonstrated to be important by activity impairment, or knockout, then conservative substitutions can be made, such as Asp for Glu to alter side chain length, Ser for Cys, or Arg for His. For hydrophobic segments, it is largely size that will be altered, although aromatics can also be substituted for alkyl side chains. Changes in the normal product distribution can indicate which step(s) of the reaction sequence have been altered by the mutation.

Other site directed mutagenesis techniques may also be employed with the nucleotide sequences of the invention. For example, restriction endonuclease digestion of DNA followed by ligation may be used to generate dirigent protein or pinoresinol/lariciresinol reductase deletion variants, as described in Section 15.3 of Sambrook et al. (Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, New York, N.Y. (1989)). A similar strategy may be used to construct insertion variants, as described in Section 15.3 of Sambrook et al., supra.

Oligonucleotide-directed mutagenesis may also be employed for preparing substitution variants of this invention. It may also be used to conveniently prepare the deletion and insertion variants of this invention. This technique is well known in the art as described by Adelman et al. (DNA 2:183 (1983)). Generally, oligonucleotides of at least 25 nucleotides in length are used to insert, delete or substitute two or more nucleotides in the dirigent protein gene or pinoresinol/lariciresinol reductase gene. An optimal oligonucleotide will have 12 to 15 perfectly matched nucleotides on either side of the nucleotides coding for the mutation. To mutagenize the wild-type dirigent protein or wild-type pinoresinol/lariciresinol reductase, the oligonucleotide is annealed to the single-stranded DNA template molecule under suitable hybridization conditions. A DNA polymerizing enzyme, usually the Klenow fragment of E. coli DNA polymerase I, is then added. This enzyme uses the oligonucleotide as a primer to complete the synthesis of the mutation-bearing strand of DNA. Thus, a heteroduplex molecule is formed such that one strand of DNA encodes the wild-type dirigent protein or pinoresinol/lariciresinol reductase inserted in the vector, and the second strand of DNA encodes the mutated form of dirigent protein or pinoresinol/lariciresinol reductase inserted into the same vector. This heteroduplex molecule is then transformed into a suitable host cell.

Mutants with more than one amino acid substituted may be generated in one of several ways. If the amino acids are located close together in the polypeptide chain, they may be mutated simultaneously using one oligonucleotide that codes for all of the desired amino acid substitutions. If however, the amino acids are located some distance from each other (separated by more than ten amino acids, for example) it is more difficult to generate a single oligonucleotide that encodes all of the desired changes. Instead, one of two alternative methods may be employed. In the first method, a separate oligonucleotide is generated for each amino acid to be substituted. The oligonucleotides are then annealed to the single-stranded template DNA simultaneously, and the second strand of DNA that is synthesized from the template will encode all of the desired amino acid substitutions.

An alternative method involves two or more rounds of mutagenesis to produce the desired mutant. The first round is as described for the single mutants: wild-type dirigent protein or pinoresinol/lariciresinol reductase DNA is used for the template, an oligonucleotide encoding the first desired amino acid substitution(s) is annealed to this template, and the heteroduplex DNA molecule is then generated. The second round of mutagenesis utilizes the mutated DNA produced in the first round of mutagenesis as the template. Thus, this template already contains one or more mutations. The oligonucleotide encoding the additional desired amino acid substitution(s) is then annealed to this template, and the resulting strand of DNA now encodes mutations from both the first and second rounds of mutagenesis. This resultant DNA can be used as a template in a third round of mutagenesis, and so on.

Eukaryotic expression systems may be utilized for dirigent protein or pinoresinol/lariciresinol reductase production since they are capable of carrying out any required posttranslational modifications and of directing the enzyme to the proper membrane location. A representative eukaryotic expression system for this purpose uses the recombinant baculovirus, Autographa californica nuclear polyhedrosis virus (AcNPV; M. D. Summers and G. E. Smith, A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures (1986); Luckow et al., Bio-technology 6:47-55 (1987)) for expression of the dirigent protein or pinoresinol/lariciresinol reductases of the invention. Infection of insect cells (such as cells of the species Spodoptera frugiperda) with the recombinant baculoviruses allows for the production of large amounts of the dirigent protein or pinoresinol/lariciresinol reductase protein. In addition, the baculovirus system has other important advantages for the production of recombinant dirigent protein or pinoresinol/lariciresinol reductase. For example, baculoviruses do not infect humans and can therefore be safely handled in large quantities. In the baculovirus system, a DNA construct is prepared including a DNA segment encoding dirigent protein or pinoresinol/lariciresinol reductase and a vector. The vector may comprise the polyhedron gene promoter region of a baculovirus, the baculovirus flanking sequences necessary for proper cross-over during recombination (the flanking sequences comprise about 200-300 base pairs adjacent to the promoter sequence) and a bacterial origin of replication which permits the construct to replicate in bacteria. The vector is constructed so that (i) the DNA segment is placed adjacent (or operably-linked or “downstream” or “under the control of”) to the polyhedron gene promoter and (ii) the promoter/pinoresinol/lariciresinol reductase, or promoter/dirigent protein, combination is flanked on both sides by 200-300 base pairs of baculovirus DNA (the flanking sequences).

To produce a dirigent protein DNA construct, or a pinoresinol/lariciresinol reductase DNA construct, a cDNA clone encoding a full length dirigent protein or pinoresinol/lariciresinol reductase is obtained using methods such as those described herein. The DNA construct is contacted in a host cell with baculovirus DNA of an appropriate baculovirus (that is, of the same species of baculovirus as the promoter encoded in the construct) under conditions such that recombination is effected. The resulting recombinant baculoviruses encode the full dirigent protein or pinoresinol/lariciresinol reductase. For example, an insect host cell can be cotransfected or transfected separately with the DNA construct and a functional baculovirus. Resulting recombinant baculoviruses can then be isolated and used to infect cells to effect production of dirigent protein or pinoresinol/lariciresinol reductase. Host insect cells include, for example, Spodoptera frugiperda cells. Insect host cells infected with a recombinant baculovirus of the present invention are then cultured under conditions allowing expression of the baculovirus-encoded dirigent protein or pinoresinol/lariciresinol reductase. Recombinant protein thus produced is then extracted from the cells using methods known in the art.

Other eukaryotic microbes such as yeasts may also be used to practice this invention. The baker's yeast Saccharomyces cerevisiae, is a commonly used yeast, although several other strains are available. The plasmid YRp7 (Stinchcomb et al., Nature 282:39 (1979); Kingsman et al., Gene 7:141 (1979); Tschemper et al., Gene 10:157 (1980)) is commonly used as an expression vector in Saccharomyces. This plasmid contains the trpl gene that provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, such as strains ATCC No. 44,076 and PEP4-1 (Jones, Genetics 85:12 (1977)). The presence of the trp1 lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan. Yeast host cells are generally transformed using the polyethylene glycol method, as described by Hinnen (Proc. Natl. Acad. Sci. USA 75:1929 (1978)). Additional yeast transformation protocols are set forth in Gietz et al., N.A.R. 20(17):1425 (1992); Reeves et al., FEMS 99:193-197 (1992).

Suitable promoting sequences in yeast vectors include the promoters for 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073 (1980)) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149 (1968); Holland et al., Biochemistry 17:4900 (1978)), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. In the construction of suitable expression plasmids, the termination sequences associated with these genes are also ligated into the expression vector 3′ of the sequence desired to be expressed to provide polyadenylation of the mRNA and termination. Other promoters that have the additional advantage of transcription controlled by growth conditions are the promoter region for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, and the aforementioned glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Any plasmid vector containing yeast-compatible promoter, origin of replication and termination sequences is suitable.

Cell cultures derived from multicellular organisms, such as plants, may be used as hosts to practice this invention. Transgenic plants can be obtained, for example, by transferring plasmids that encode pinoresinol/lariciresinol reductase, and/or dirigent protein, and a selectable marker gene, e.g., the kan gene encoding resistance to kanamycin, into Agrobacterium tumifaciens containing a helper Ti plasmid as described in Hoeckema et al., Nature 303:179-181 (1983) and culturing the Agrobacterium cells with leaf slices of the plant to be transformed as described by An et al., Plant Physiology 81:301-305 (1986). Transformation of cultured plant host cells is normally accomplished through Agrobacterium tumifaciens, as described above. Cultures of mammalian host cells and other host cells that do not have rigid cell membrane barriers are usually transformed using the calcium phosphate method as originally described by Graham and Van der Eb (Virology 52:546 (1978)) and modified as described in Sections 16.32-16.37 of Sambrook et al., supra. However, other methods for introducing DNA into cells such as Polybrene (Kawai and Nishizawa, Mol. Cell. Bio l. 4:1172 (1984)), protoplast fusion (Schaffner, Proc. Natl. Acad. Sci. USA 77:2163 (1980)), electroporation (Neumann et al., EMBO J. 1:841 (1982)), and direct microinjection into nuclei (Capecchi, Cell 22:479 (1980)) may also be used. Additionally, animal transformation strategies are reviewed in Monastersky G. M. and Robl, J. M., Strategies in Transgenic Animal Science, ASM Press, Washington, D.C. (1995). Transformed plant calli may be selected through the selectable marker by growing the cells on a medium containing, e.g., kanamycin, and appropriate amounts of phytohormone such as naphthalene acetic acid and benzyladenine for callus and shoot induction. The plant cells may then be regenerated and the resulting plants transferred to soil using techniques well known to those skilled in the art.

In addition, a gene regulating pinoresinol/lariciresinol reductase production, or dirigent protein production, can be incorporated into the plant along with a necessary promoter which is inducible. In the practice of this embodiment of the invention, a promoter that only responds to a specific external or internal stimulus is fused to the target cDNA. Thus, the gene will not be transcribed except in response to the specific stimulus. As long as the gene is not being transcribed, its gene product is not produced.

An illustrative example of a responsive promoter system that can be used in the practice of this invention is the glutathione-S-transferase (GST) system in maize. GSTs are a family of enzymes that can detoxify a number of hydrophobic electrophilic compounds that often are used as pre-emergent herbicides (Weigand et al., Plant Molecular Biology 7:235-243 (1986)). Studies have shown that the GSTs are directly involved in causing this enhanced herbicide tolerance. This action is primarily mediated through a specific 1.1 kb mRNA transcription product. In short, maize has a naturally occurring quiescent gene already present that can respond to external stimuli and that can be induced to produce a gene product. This gene has previously been identified and cloned. Thus, in one embodiment of this invention, the promoter is removed from the GST responsive gene and attached to a pinoresinol/lariciresinol reductase gene, or a dirigent protein gene, that previously has had its native promoter removed. This engineered gene is the combination of a promoter that responds to an external chemical stimulus and a gene responsible for successful production of pinoresinol/lariciresinol reductase or dirigent protein.

In addition to the methods described above, several methods are known in the art for transferring cloned DNA into a wide variety of plant species, including gymnosperms, angiosperms, monocots and dicots (see, e.g., Glick and Thompson, eds., Methods in Plant Molecular Biology, CRC Press, Boca Raton, Fla. (1993)). Representative examples include electroporation-facilitated DNA uptake by protoplasts (Rhodes et al., Science 240(4849):204-207 (1988)); treatment of protoplasts with polyethylene glycol (Lyznik et al., Plant Molecular Biology 13:151-161 (1989)); and bombardment of cells with DNA laden microprojectiles (Klein et al., Plant Physiol. 91:440-444 (1989) and Boynton et al., Science 240(4858):1534-1538 (1988)). Numerous methods now exist, for example, for the transformation of cereal crops (see, e.g., McKinnon, G. E. and Henry, R. J., J. Cereal Science, 22(3):203-210 (1995); Mendel, R. R. and Teeri, T. H., Plant and Microbial Biotechnology Research Series, 3:81-98, Cambridge University Press (1995); McElroy, D. and Brettell, R. I. S., Trends in Biotechnology, 12(2):62-68 (1994); Christou et al., Trends in Biotechnology, 10(7):239-246 (1992); Christou, P. and Ford, T. L., Annals of Botany, 75(5): 449-454 (1995); Park et al., Plant Molecular Biology, 32(6):1135-1148 (1996); Altpeter et al., Plant Cell Reports, 16:12-17 (1996)). Additionally, plant transformation strategies and techniques are reviewed in Birch, R. G., Ann Rev Plant Phys Plant Mol Biol 48:297 (1997); Forester et al., Exp. Agric. 33:15-33 (1997). Minor variations make these technologies applicable to a broad range of plant species.

Each of these techniques has advantages and disadvantages. In each of the techniques, DNA from a plasmid is genetically engineered such that it contains not only the gene of interest, but also selectable and screenable marker genes. A selectable marker gene is used to select only those cells that have integrated copies of the plasmid (the construction is such that the gene of interest and the selectable and screenable genes are transferred as a unit). The screenable gene provides another check for the successful culturing of only those cells carrying the genes of interest. A commonly used selectable marker gene is neomycin phosphotransferase II (NPT II). This gene conveys resistance to kanamycin, a compound that can be added directly to the growth media on which the cells grow. Plant cells are normally susceptible to kanamycin and, as a result, die. The presence of the NPT II gene overcomes the effects of the kanamycin and each cell with this gene remains viable. Another selectable marker gene which can be employed in the practice of this invention is the gene which confers resistance to the herbicide glufosinate (Basta). A screenable gene commonly used is the β-glucuronidase gene (GUS). The presence of this gene is characterized using a histochemical reaction in which a sample of putatively transformed cells is treated with a GUS assay solution. After an appropriate incubation, the cells containing the GUS gene turn blue. Preferably, the plasmid will contain both selectable and screenable marker genes.

The plasmid containing one or more of these genes is introduced into either plant protoplasts or callus cells by any of the previously mentioned techniques. If the marker gene is a selectable gene, only those cells that have incorporated the DNA package survive under selection with the appropriate phytotoxic agent. Once the appropriate cells are identified and propagated, plants are regenerated. Progeny from the transformed plants must be tested to insure that the DNA package has been successfully integrated into the plant genome.

Mammalian host cells may also be used in the practice of the invention. Examples of suitable mammalian cell lines include monkey kidney CVI line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line 293S (Graham et al., J. Gen. Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (Urlab and Chasin, Proc. Natl. Acad. Sci USA 77:4216 (1980)); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243 (1980)); monkey kidney cells (CVI-76, ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor cells (MMT 060562, ATCC CCL 51); rat hepatoma cells (HTC, MI.54, Baumann et al., J. Cell Biol. 85:1 (1980)); and TRI cells (Mather et al., Annals N.Y. Acad. Sci. 383:44 (1982)). Expression vectors for these cells ordinarily include (if necessary) DNA sequences for an origin of replication, a promoter located in front of the gene to be expressed, a ribosome binding site, an RNA splice site, a polyadenylation site, and a transcription terminator site.

Promoters used in mammalian expression vectors are often of viral origin. These viral promoters are commonly derived from polyoma virus, Adenovirus 2, and most frequently Simian Virus 40 (SV40). The SV40 virus contains two promoters that are termed the early and late promoters. These promoters are particularly useful because they are both easily obtained from the virus as one DNA fragment that also contains the viral origin of replication (Fiers et al., Nature 273:113 (1978)). Smaller or larger SV40 DNA fragments may also be used, provided they contain the approximately 250-bp sequence extending from the HindlIl site toward the BglI site located in the viral origin of replication.

Alternatively, promoters that are naturally associated with the foreign gene (homologous promoters) may be used provided that they are compatible with the host cell line selected for transformation.

An origin of replication may be obtained from an exogenous source, such as SV40 or other virus (e.g., Polyoma, Adeno, VSV, BPV) and inserted into the cloning vector. Alternatively, the origin of replication may be provided by the host cell chromosomal replication mechanism. If the vector containing the foreign gene is integrated into the host cell chromosome, the latter is often sufficient.

The use of a secondary DNA coding sequence can enhance production levels of pinoresinol/lariciresinol reductase or dirigent protein in transformed cell lines. The secondary coding sequence typically comprises the enzyme dihydrofolate reductase (DHFR). The wild-type form of DHFR is normally inhibited by the chemical methotrexate (MTX). The level of DHFR expression in a cell will vary depending on the amount of MTX added to the cultured host cells. An additional feature of DHFR that makes it particularly useful as a secondary sequence is that it can be used as a selection marker to identify transformed cells. Two forms of DHFR are available for use as secondary sequences, wild-type DHFR and MTX-resistant DHFR. The type of DHFR used in a particular host cell depends on whether the host cell is DHFR deficient (such that it either produces very low levels of DHFR endogenously, or it does not produce functional DHFR at all). DHFR-deficient cell lines such as the CHO cell line described by Urlaub and Chasin, supra, are transformed with wild-type DHFR coding sequences. After transformation, these DHFR-deficient cell lines express functional DHFR and are capable of growing in a culture medium lacking the nutrients hypoxanthine, glycine and thymidine. Nontransformed cells will not survive in this medium.

The MTX-resistant form of DHFR can be used as a means of selecting for transformed host cells in those host cells that endogenously produce normal amounts of functional DHFR that is MTX sensitive. The CHO-Kl cell line (ATCC No. CL 61) possesses these characteristics, and is thus a useful cell line for this purpose. The addition of MTX to the cell culture medium will permit only those cells transformed with the DNA encoding the MTX-resistant DHFR to grow. The nontransformed cells will be unable to survive in this medium.

Prokaryotes may also be used as host cells for the initial cloning steps of this invention. They are particularly useful for rapid production of large amounts of DNA, for production of single-stranded DNA templates used for site-directed mutagenesis, for screening many mutants simultaneously, and for DNA sequencing of the mutants generated. Suitable prokaryotic host cells include E. coli K12 strain 294 (ATCC No. 31,446), E. coli strain W3110 (ATCC No.27,325) E. coli X1776 (ATCC No. 31,537), and E. coli B; however many other strains of E. coli, such as HB101, JM101, NM522, NM538, NM539, and many other species and genera of prokaryotes including bacilli such as Bacillus subtilis, other enterobacteriaceae such as Salmonella typhimurium or Serratia marcesans, and various Pseudomonas species may all be used as hosts. Prokaryotic host cells or other host cells with rigid cell walls are preferably transformed using the calcium chloride method as described in section 1.82 of Sambrook et al., supra. Alternatively, electroporation may be used for transformation of these cells. Prokaryote transformation techniques are set forth in Dower, W. J., in Genetic Engineering, Principles and Methods, 12:275-296, Plenum Publishing Corp. (1990); Hanahan et al., Meth. Enxymol., 204:63 (1991).

As a representative example, cDNA sequences encoding dirigent protein or pinoresinol/lariciresinol reductase may be transferred to the (His)₆·Tag pET vector commercially available (from Novagen) for overexpression in E. coli as heterologous host. This pET expression plasmid has several advantages in high level heterologous expression systems. The desired cDNA insert is ligated in frame to plasmid vector sequences encoding six histidines followed by a highly specific protease recognition site (thrombin) that are joined to the amino terminus codon of the target protein. The histidine “block” of the expressed fusion protein promotes very tight binding to immobilized metal ions and permits rapid purification of the recombinant protein by immobilized metal ion affinity chromatography. The histidine leader sequence is then cleaved at the specific proteolysis site by treatment of the purified protein with thrombin, and the dirigent protein or pinoresinol/lariciresinol reductase eluted. This overexpression-purification system has high capacity, excellent resolving power and is fast, and the chance of a contaminating E. coli protein exhibiting similar binding behavior (before and after thrombin proteolysis) is extremely small.

As will be apparent to those skilled in the art, any plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell may also be used in the practice of the invention. The vector usually has a replication site, marker genes that provide phenotypic selection in transformed cells, one or more promoters, and a polylinker region containing several restriction sites for insertion of foreign DNA. Plasmids typically used for transformation of E. coli include pBR322, pUC18, pUC19, pUCI18, pUC119, and Bluescript M13, all of which are described in Sections 1.12-1.20 of Sambrook et al., supra. However, many other suitable vectors are available as well. These vectors contain genes coding for ampicillin and/or tetracycline resistance which enables cells transformed with these vectors to grow in the presence of these antibiotics.

The promoters most commonly used in prokaryotic vectors include the β-lactamase (penicillinase) and lactose promoter systems (Chang et al. Nature 375:615 (1978); Itakura et al., Science 198:1056 (1977); Goeddel et al., Nature 281:544 (1979)) and a tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057 (1980); EPO Appl. Publ. No. 36,776), and the alkaline phosphatase systems. While these are the most commonly used, other microbial promoters have been utilized, and details concerning their nucleotide sequences have been published, enabling a skilled worker to ligate them functionally into plasmid vectors (see Siebenlist et al., Cell 20:269 (1980)).

Many eukaryotic proteins normally secreted from the cell contain an endogenous secretion signal sequence as part of the amino acid sequence. Thus, proteins normally found in the cytoplasm can be targeted for secretion by linking a signal sequence to the protein. This is readily accomplished by ligating DNA encoding a signal sequence to the 5′ end of the DNA encoding the protein and then expressing this fusion protein in an appropriate host cell. The DNA encoding the signal sequence may be obtained as a restriction fragment from any gene encoding a protein with a signal sequence. Thus, prokaryotic, yeast, and eukaryotic signal sequences may be used herein, depending on the type of host cell utilized to practice the invention. The DNA and amino acid sequence encoding the signal sequence portion of several eukaryotic genes including, for example, human growth hormone, proinsulin, and proalbumin are known (see Stryer, Biochemistry W.H. Freeman and Company, New York, N.Y., p. 769 (1988)), and can be used as signal sequences in appropriate eukaryotic host cells. Yeast signal sequences, as for example acid phosphatase (Arima et al., Nucleic Acids Res. 11:1657 (1983)), alpha-factor, alkaline phosphatase and invertase may be used to direct secretion from yeast host cells. Prokaryotic signal sequences from genes encoding, for example, LamB or OmpF (Wong et al., Gene 68:193 (1988)), MalE, PhoA, or beta-lactamase, as well as other genes, may be used to target proteins from prokaryotic cells into the culture medium.

Trafficking sequences from plants, animals and microbes can be employed in the practice of the invention to direct the gene product to the cytoplasm, endoplasmic reticulum, mitochondria or other cellular components, or to target the protein for export to the medium. These considerations apply to the overexpression of pinoresinol/lariciresinol reductase or dirigent protein, and to direction of expression within cells or intact organisms to permit gene product function in any desired location.

The construction of suitable vectors containing DNA encoding replication sequences, regulatory sequences, phenotypic selection genes and the dirigent protein DNA or pinoresinol/lariciresinol reductase DNA of interest are prepared using standard recombinant DNA procedures. Isolated plasmids and DNA fragments are cleaved, tailored, and ligated together in a specific order to generate the desired vectors, as is well known in the art (see, for example, Sambrook et al., supra).

As discussed above, pinoresinol/lariciresinol reductase variants, or dirigent protein variants, are preferably produced by means of mutation(s) that are generated using the method of site-specific mutagenesis. This method requires the synthesis and use of specific oligonucleotides that encode both the sequence of the desired mutation and a sufficient number of adjacent nucleotides to allow the oligonucleotide to stably hybridize to the DNA template.

A dirigent protein gene and/or pinoresinol/lariciresinol reductase gene, or an antisense nucleic acid fragment complementary to all or part of a dirigent protein gene or pinoresinol/lariciresinol reductase gene, may be introduced, as appropriate, into any plant species for a variety of purposes including, but not limited to: altering or improving the color, texture, durability and pest-resistance of wood tissue, especially heartwood tissue; reducing the formation of lignans and/or lignins in plant species, such as corn, which are useful as animal fodder, thereby enhancing the availability of the cellulose fraction of the plant material to the digestive system of animals ingesting the plant material; reducing the lignan/lignin content of plant species utilized in pulp and paper production, thereby making pulp and paper production easier and cheaper; improving the defensive capability of a plant against predators and pathogens by enhancing the production of defensive lignans or lignins; the alteration of other ecological interactions mediated by lignans or lignins; producing elevated levels of optically-pure lignan enantiomers as medicines or food additives; introducing, enhancing or inhibiting the production of dirigent proteins or pinoresinol/lariciresinol reductases, or the production of pinoresinol or lariciresinol and their derivatives. A dirigent protein and/or pinoresinol/lariciresinol reductase gene may be introduced into any organism for a variety of purposes including, but not limited to: introducing, enhancing or inhibiting the production of dirigent protein and/or pinoresinol/lariciresinol reductase, or the production of pinoresinol or lariciresinol and their derivatives.

The foregoing may be more fully understood in connection with the following representative examples, in which “Plasmids” are designated by a lower case p followed by an alphanumeric designation. The starting plasmids used in this invention are either commercially available, publicly available on an unrestricted basis, or can be constructed from such available plasmids using published procedures. In addition, other equivalent plasmids are known in the art and will be apparent to the ordinary artisan.

“Digestion”, “cutting” or “cleaving” of DNA refers to catalytic cleavage of the DNA with an enzyme that acts only at particular locations in the DNA. These enzymes are called restriction endonucleases, and the site along the DNA sequence where each enzyme cleaves is called a restriction site. The restriction enzymes used in this invention are commercially available and are used according to the instructions supplied by the manufacturers. (See also Sections 1.60-1.61 and Sections 3.38-3.39 of Sambrook et al., supra.)

“Recovery” or “isolation” of a given fragment of DNA from a restriction digest means separation of the resulting DNA fragment on a polyacrylamide or an agarose gel by electrophoresis, identification of the fragment of interest by comparison of its mobility versus that of marker DNA fragments of known molecular weight, removal of the gel section containing the desired fragment, and separation of the gel from DNA. This procedure is known generally. For example, see Lawn et al. (Nucleic Acids Res. 9:6103-6114 (1982)), and Goeddel et al. (Nucleic Acids Res., supra).

The following examples merely illustrate the best mode now contemplated for practicing the invention, but should not be construed to limit the invention. All literature citations herein are expressly incorporated by reference.

EXAMPLE 1 Purification of Dirigent Protein from Forsythia intermedia

Plant Materials. Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, Minn.), and maintained in Washington State University greenhouse facilities, or were gifts from the local community.

Initial Extraction and Ammonium Sulphate Precipitation. Solubilization of bound proteins was carried out at 4° C. Frozen Forsythia intermedia stems (2 kg) were pulverized in a Waring Blendor (Model CB6) in the presence of liquid nitrogen. The resulting powder was homogenized with 0.1 M KH₂PO₄-K₂HPO₄ buffer (pH 7.0, 4 liters) containing 5 mM dithiothreitol, and filtered through four layers of cheesecloth. The insoluble residue was consecutively extracted, with continuous agitation at 250 rpm, as follows: with chilled (−20° C.) re-distilled acetone (4 liters, 3×30 min); 0.1 M KH₂PO₄-K₂HPO₄ buffer (pH 6.5) containing 0.1% β-mercaptoethanol (solution A, 8 liters, 30 min); solution A containing 1% Triton X100 (8 liters, 4 hours) and finally solution A (8 liters, 16 hours). Between each extraction, the residue was filtered through one layer of Miracloth (Calbiochem). Solubilization of the (+)-pinoresinol forming system was achieved by mechanically stirring the residue in solution A containing 1 M NaCl (8 liters, 4 hours). The homogenate was decanted and the resulting solution consecutively filtered through Miracloth (Calbiochem) and glass fiber (G6, Fisher Sci.). The filtrate was concentrated in an Amicon cell (Model 2000, YM 30 membrane) to a final volume of ˜800 ml, and subjected to (NH₄)₂SO₄ fractionation. Proteins precipitating between 40 and 80% saturation were recovered by centrifugation (15,000 g, 30 min) and the (NH₄)₂SO₄ pellet stored at −20° C. until required.

Mono S Column Chromatography. Purification of 78-kD dirigent protein and partial purification of oxidase. The ammonium sulfate pellet (obtained from 2 kg of F intermedia stems) was reconstituted in 40 mM MES [2-(N-Morpholino)ethanesulfonic acid] buffer, adjusted to pH 5.0 with 6 M NaOH (solution B, 30 ml), the slurry being centrifuged (3,600 g, 5 min), and the supernatant dialyzed overnight against solution B (4 liters). The dialyzed extract was filtered (0.22 μm) and the sample (35 to 40 mg proteins) was applied to a MonoS HR5/5 (50 mm by 5 mm) column equilibrated in solution B at 4° C. After eluting (flow rate 5 ml min⁻¹ cm⁻²) with solution B (13 ml), proteins were desorbed with Na₂SO₄ in solution B, using a linear gradient from 0 to 100 mM in 8 ml and holding at this concentration for 32 ml, then implementing a series of step gradients at 133 mM for 50 ml, 166 mM for 50 ml, 200 mM for 40 ml, 233 mM for 40 ml and finally 333 mM Na₂SO₄ for 40 ml. Fractions capable of forming (+)-pinoresinol from E-coniferyl alcohol were eluted with 333 mM Na₂SO₄, combined and stored (−80° C.) until needed.

POROS SP-M Matrix Column Chromatography (First Column). Fractions from 15 individual elutions from the MonoS HR5/5 column (33 mM Na₂SO₄) were combined (18.5 mg proteins, 180 ml) and dialyzed overnight against solution C. The dialyzed enzyme solution (190 ml) was filtered (0.22 μm) and an aliquot (47 ml) was applied to the POROS SP-M column. All separations on a POROS SP-M matrix (100 mm by 4.6 mm), previously equilibrated in 25 mM MES-HEPES-sodium acetate buffer (pH 5.0, solution C), were performed at a flow rate of 60 ml min⁻¹ cm⁻² and at room temperature. After elution with solution C (12 ml), the proteins were desorbed with a linear Na₂SO₄ gradient (0 to 0.7 M in 66.5 ml) in solution C, whereupon the concentration established was held for an additional 16.6 ml. Under these conditions, separation of four fractions (I, II, III and IV) was achieved at ˜40, 47, 55 and 61 mS, respectively. This purification step was repeated three times with the remaining dialyzed enzymatic extract, and fractions I, II, III, and IV from each experiment were separately combined. When protease inhibitors [that is, phenylmethanesulfonyl fluoride (0.1 mmol ml⁻¹), EDTA (0.5 nmol ml⁻¹), pepstatin A (1 μg ml⁻¹), and antipain (1 μg ml⁻¹)] were added during the solubilization and all subsequent purification stages, no differences were observed in the elution profiles of fractions I, II, III, and IV.

POROS SP-M Matrix Column Chromatography (Second Column). Fraction I from the first POROS SP-M Matrix column chromatography step (2.62 mg proteins, 40 ml, ˜24.6 mS) was diluted in filtered, cold distilled water until the conductivity reached ˜8 mS (final volume=150 ml). The diluted protein solution was then applied onto a POROS SP-M column (100 mm by 4.6 mm). After elution with solution C (12 ml), fraction I was desorbed using a linear Na₂SO₄ gradient from 0 to 0.25 M in 20 ml, whereupon the concentration established was held for another 25 ml. This was followed by another linear Na₂SO₄ gradient from 0.25 to 0.7 M in 26 ml which was then held at 0.7 M for an additional 16.6 ml. Fractions eluted at ˜30 mS (the ionic strength of the eluent was measured with a flow-through detector) were combined (15 ml, 1.3 mg), diluted with water and rechromatographed. The resulting protein (eluted at ˜30 mS with the gradient described above) was stored (−80° C.) until needed.

Gel filtration. An aliquot from fraction I (595.5 μg proteins, 3 ml, eluted at ˜30 mS), was concentrated to 0.6 ml (Centricon 10, Amicon) and loaded onto a S200 (73.2 cm by 1.6 cm, Pharmacia-LKB) gel chromatographic column equilibrated in 0.1 M MES-HEPES-sodium acetate buffer (pH 5.0) containing 50 mM Na₂SO₄ at 4° C. An apparently homogenous 78-kD dirigent protein (242 μg) was eluted (flow rate 0.25 ml min⁻¹ cm⁻²) as a single component at 133 ml (Vo=105 ml). Molecular weights were estimated by comparison of their elution profiles with the standard proteins, β-amylase (200,000), alcohol dehydrogenase (150,000), bovine serum albumin (66,000), ovalbumin (45,000), carbonic anhydrase (29,000) and cytochrome c (12,400).

EXAMPLE 2 Characterization of the Purified Dirigent Protein

Molecular Weight and Isoelectric Point Determination. Polyacrylamide gel electrophoresis (PAGE) was performed in Laemmli's buffer system with gradient (4 to 15% acrylamide, Bio-Rad) gels under denaturing and reducing conditions. Proteins were visualized by silver staining. Gel filtration (S200) chromatography of fraction I gave a protein of native molecular weight ˜78 kD, whereas SDS-polyacrylamide gel electrophoresis showed a single band at ˜27 kD, suggesting that the native protein exists as a trimer. Isoelectric focusing of the native protein on a polyacrylamide gel (pH 3 to 10 gradient) revealed the presence of six bands. After isoelectric focusing, each of these bands was electroblotted onto a polyvinylidene fluoride (PVDF) membrane and subjected to amino terminal sequencing, which established that all had similar sequences indicating a series of isoforms. The ultraviolet-visible spectrum of the protein had only a characteristic protein absorbance at 280 nm with a barely perceptible shoulder at ˜330 nm. Inductively coupled plasma (ICP) analysis gave no indication of any metal being present in the protein. Thus, the 78-kD dirigent protein lacks any detectable catalytically active oxidative center.

Assay of the Ability of the Purified Dirigent Protein to Form (+)Pinoresinol from E-Coniferyl alcohol. The four fractions (I to IV) from the first POROS SP-M chromatographic step (Example 1) were individually rechromatographed, with each fraction subsequently assayed for (+)-pinoresinol-forming activity with E-[9-³H]coniferyl alcohol as substrate for one hour. Fraction I (containing dirigent protein) had very little (+)-pinoresinol-forming activity (<5% of total activity loaded onto the POROS SP-M column), whereas fraction III catalyzed nonspecific oxidative coupling to give (±)-dehydrodiconiferyl alcohols, (±)-pinoresinols, and (±)-erythro/threo guaiacylglycerol 8-O-4′-coniferyl alcohol ethers. Thus, Fraction III appeared to contain an endogenous plant oxygenating protein.

Although the putative oxidase preparation (Fraction III) was not purified to electrophoretic homogeneity, the electron paramagnetic resonance (EPR) spectrum of this protein preparation resembled that of a typical plant laccase, i.e., a class of naturally-occurring plant oxygenase proteins. We then studied the fate of E-[9-³H]coniferyl alcohol (2 μmol ml⁻¹, 14.7 kBq) in the presence of, respectively, the oxidase (fraction III), the 78-kD dirigent protein (Fraction I), and both fraction III and the 78-kD protein together. With the fraction III preparation alone, only nonspecific bimolecular radical coupling occurs to give (±)-dehydrodiconiferyl alcohols, (±)-pinoresinols and (±)-erythro/threo guaiacylglycerol 8-O-4′-coniferyl alcohol ethers. With the 78-kD protein by itself, however, a small amount of (+)-pinoresinol formation (<5% over 10 hours) was observed, this being presumed to result from residual traces of oxidizing capacity in the preparation. When both fraction III and the 78-kD protein were combined, full catalytic activity and regio- and stereo-specificity in the product was reestablished, whereby essentially only (+)-pinoresinol was formed. Additionally, with fraction III alone, and when fraction III was combined with the 78-kD protein, the rates of substrate depletion and dimeric product formation were nearly identical. Moreover, essentially no turnover of the dimeric lignan products occurred in either case in the presence of the oxidase, over the time-period (8 hours) examined: subsequent dimer oxidation does not occur when E-coniferyl alcohol, the preferred substrate, is still present in the assay mixture. The 78-kD protein therefore appears to determine the specificity of the bimolecular phenoxy radical coupling reaction.

Gel filtration studies were also carried out with mixtures of the dirigent and fraction III proteins, in order to establish if any detectable protein-protein interaction might account for the stereoselectivity. But no evidence in support of complex formation (i.e., to higher molecular size entities) was observed.

EXAMPLE 3 Effect of the 78-KD Dirigent Protein on Plant Laccase-Catalyzed Monolignol Coupling

E-coniferyl alcohol coupling assay. E-[9-³H]Coniferyl alcohol (4 μmol ml⁻¹, 29.3 kBq) was incubated with a 120-kD laccase (previously purified from Forsythia intermedia stem tissue) over a 24-hour period, in the presence and absence of the dirigent protein, as follows. Each assay consisted of E-[9-³H]coniferyl alcohol (4 μmol ml⁻¹, 29.3 kBq, 7.3 MBq mole liter⁻¹; or 2 μmol ml⁻¹, 14.7 kBq with fraction III), the 78-kD dirigent protein, an oxidase or oxidant, or both [final concentrations: 770 pmol ml⁻¹ dirigent protein; 10.7 pmol protein ml⁻¹ Forsythia laccase; 12 μg protein ml⁻¹ fraction III; 0.5 μmol ml⁻¹ FMN; 0.5 μmol ml⁻¹ FAD; 1 and 10 μmol ml⁻¹ ammonium peroxydisulfate] in buffer (0.1 M MES-HEPES-sodium acetate, pH 5.0) to a total volume of 250 μl. The enzymatic reaction was initiated by addition of E-[9-³H]coniferyl alcohol. Controls were performed in the presence of buffer alone.

After one hour incubation at 30° C. while shaking, the assay mixture was extracted with ethyl acetate (EtOAc, 500 μl) containing (±)-pinoresinols (7.5 μg), (±)-dehydrodiconiferyl alcohols (3.5 μg) and erythro/threo (±)-guaiacylglycerol 8-O-4-coniferyl alcohol ethers (7.5 μg) as radiochemical carriers and ferulic acid (15.0 μg) as an internal standard. After centrifugation (13,800 g, 5 min), the EtOAc soluble components were removed and the extraction procedure repeated with EtOAc (500 μl). The EtOAc soluble components from each assay were combined, the solutions evaporated to dryness in vacuo, redissolved in methanol-water solution (1:1; 100 μl) with an aliquot (50 μl) thereof subjected to reversed-phase column chromatography (Waters, Nova-Pak C₁₈, 150 mm by 3.8 mm). The elution conditions were as follows: acetonitrile/3% acetic acid in H₂O (5:95) from 0 to 5 min, then linear gradients to ratios of 10:90 between 5 and 20 min, then to 20:80 between 20 and 45 min and finally to 50:50 between 45 and 60 min, at a flow rate of 8.8 ml min⁻¹ cm⁻².

Fractions corresponding to E-coniferyl alcohol, erythro/threo (±)-guaiacylglycerol 8-O-4′-coniferyl alcohol ethers, (±)-dehydrodiconiferyl alcohols and (±)-pinoresinols were individually collected, aliquots removed for liquid scintillation counting, and the remainder freeze-dried. Pinoresinol-containing fractions were redissolved in methanol (100 μl) and subjected to chiral column chromatography (Daicel, Chiralcel OD, 50 mm by 4.6 mm) with a solution of hexanes and ethanol (1:1) as the mobile phase (flow rate 3 ml min⁻¹ cm⁻²), whereas dehydrodiconiferyl alcohol fractions were subjected to Chiralcel OF (250 mm by 4.6 mm) column chromatography eluted with a solution of hexanes and isopropanol (9:1) as the mobile phase (flow rate 2.4 ml min⁻¹ cm⁻²), the radioactivity of the eluent being measured with a flow-through detector (Radiomatic, Model A120).

Results of E-coniferyl alcohol coupling assay. Incubation with laccase alone gave only racemic dimeric products, with (±)-dehydrodiconiferyl alcohols predominating. In the presence of the dirigent protein, however, the process was now primarily stereoselective, affording (+)-pinoresinol, rather than being nonspecific as observed when only laccase was present. The rates of both E-coniferyl alcohol (substrate) depletion and the formation of the dimeric lignans were similar with and without the dirigent protein. A substantial difference was noted in the subsequent turnover of the lignan products observed after E-coniferyl alcohol depletion. With the laccase alone no turnover occurred, but when both proteins were present the disappearance of the products was significant. In order to understand the difference, assays were conducted where bovine serum albumin (BSA) and ovalbumin were individually added to the laccase-containing solutions at levels matching the weight concentrations of the dirigent protein. In this way, it was established that the differences in product turnover were simply due to stabilization of laccase activity at the higher protein concentrations, although interestingly the dirigent protein, BSA and ovalbumin afforded somewhat different degrees of protection. The findings were quite comparable when a fungal laccase (from Trametes versicolor) was used in place of the plant laccase. When the oxidizing capacity (i.e., laccase concentration) was lowered five-fold, only (+)-pinoresinol formation was observed. Thus, complete stereoselectivity is preserved when the oxidative capacity does not exceed a point where the dirigent protein is saturated.

Stereoselective E-coniferyl alcohol coupling Assays were also conducted with E-[9-²H₂, OC²H₃]coniferyl alcohol and the dirigent protein in the presence of laccase as follows. E-[9-²H₂, OC²H₃]coniferyl alcohol (2 μmol ml⁻¹) was incubated in the presence of dirigent protein (770 pmol ml⁻¹), the purified plant laccase (4.1 pmol ml⁻¹) and buffer (0.1 M MES-HEPES-sodium acetate, pH 5.0) in a total volume of 250 μl. After one hour incubation, the reaction mixture was extracted with EtOAc, but with the addition of an internal standard and radiochemical carriers omitted. After reversed-phase column chromatography, the enzymatically formed pinoresinol was collected, freeze-dried, redissolved in methanol (100 μl) and subjected to chiral column chromatography (Daicel, Chiralcel OD, 50 mm by 4.6 mm) with detection at 280 nm and analysis by mass spectral fragmentation in the EI mode (Waters, Integrity System). Liquid chromatography-mass spectrometry (LC-MS) analysis of the resulting (+)-pinoresinol (>99% enantiomeric excess) gave a molecular ion with a mass to charge ratio (m/z) 368, thus establishing the presence of 10 ²H atoms and verifying that together the laccase and dirigent protein catalyzed stereoselective coupling of E-[9-²H₂, OC²H₃]coniferyl alcohol.

Other auxiliary one-electron oxidants can also facilitate stereoselective coupling with the dirigent protein. Ammonium peroxydisulfate readily undergoes homolytic cleavage (A. Usaitis, R. Makuska, Polymer 35:4896 (1994)) and is routinely used as an one-electron oxidant in acrylamide polymerization. Ammonium peroxydisulfate was first incubated with E-[9-³H]coniferyl alcohol (4 μmol ml⁻¹, 29.3 kBq) for 6 hours using the E-coniferyl alcohol coupling assay procedure described above. Nonspecific bimolecular radical coupling was observed, to afford predominantly (±)-dehydrodiconiferyl alcohols as well as the other racemic lignans (Table 1). However, when the dirigent protein was added, the stereoselectivity of coupling was dramatically altered to give primarily (+)-pinoresinol at both concentrations of oxidant, together with small amounts of racemic lignans. This established that even an inorganic oxidant, such as ammonium peroxydisulfate, could promote (+)-pinoresinol synthesis in the presence of the dirigent protein, even if it was not oxidatively as selective toward the monolignol as was the fraction III oxidase or laccase.

TABLE 1 Effect of dirigent protein on product distribution from E-coniferyl alcohol oxidized by ammonium peroxydisulfate (6 hour assay). E-Coniferyl alcohol (±)-Guaiacyl-glycerol (±)-Dehydro- in dimer equivalents 8-O-4′-coniferyl diconiferyl Dirigent protein depleted alcohol ethers alcohols (±)-Pinoresinols (+)-Pinoresinol Total dimers Oxidant (770 pmol ml⁻¹) (nmol ml⁻¹) (nmol ml⁻¹) (nmol ml⁻¹) (nmol ml⁻¹) (nmol ml⁻¹) (nmol ml⁻¹) Ammonium absent 200 ± 4  10 ± 1 35 ± 2 16 ± 0 0 61 ± 3 peroxydisulfate (1 μmol ml⁻¹) present 250 ± 55  6 ± 0 13 ± 1 0 130 ± 10 149 ± 11 Ammonium absent 860 ± 30 90 ± 4 250 ± 10 135 ± 4  0 475 ± 17 peroxydisulfate (10 μmol ml⁻¹) present 1030 ± 25  30 ± 1 90 ± 3 0 450 ± 10 570 ± 14 Dirigent protein present  61 ± 20  5 ± 1  8 ± 1 0 55 ± 1 68 ± 3

Effect of Other Oxygenating Agents on the Stereospecific Conversion of E-Coniferyl Alcohol to (+)-pinoresinol. The effects of incubating E-coniferyl alcohol (4 μmol ml⁻¹, 29.3 kBq) with flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) were investigated since, in addition to their roles as enzyme cofactors, they can also oxidize various organic substrates (T. C. Bruice, Acc. Chem. Res. 13:256 (1980)). E-[9-³H]coniferyl alcohol was respectively incubated with FMN and FAD for 48 hours. To obtain the FMN, snake (Naja naja atra, Formosan cobra) venom was added to a solution of FAD (5 μmol ml⁻¹ in H₂O) and, after 30 min incubation at 30° C., the enzymatically formed FMN was separated from the protein mixture by filtration through a Centricon 10 (Amicon) microconcentrator. In every instance, E-coniferyl alcohol oxidation was more rapid in the presence of FMN than FAD. Although these differences between the FMN and FAD catalyzed rates of E-coniferyl alcohol oxidation were not anticipated, a consistent pattern was sustained: racemic lignan products were obtained, with the (±)-dehydrodiconiferyl alcohols predominating as before. When the time courses were repeated in the presence of the dirigent protein, a dramatic change in stereoselectivity was observed, where essentially only (+)-pinoresinol formation occurred. Again, the rates of E-coniferyl alcohol depletion, when adjusted for the traces of residual oxidizing capacity (<5% over 10 hours) in the dirigent protein preparation, were dependent only upon [FMN] and [FAD], as were the total amounts of dimers formed. When full depletion of E-coniferyl alcohol occurs, the corresponding lignan dimers can begin to undergo oxidative changes as a function of time; specifically, FMN is able subsequently to oxidize pinoresinol, in open solution, after the E-coniferyl alcohol has been fully depleted.

Investigation of Substrate-Specific Stereoselectivity. The coupling stereoselectivity was substrate specific. Neither E-p-[9-³H]coumaryl (4 μmol ml⁻¹, 44.5 kBq) or E-[8-¹⁴C]sinapyl alcohols (4 μmol ml⁻¹, 8.3 kBq), which differ from E-coniferyl alcohol only by a methoxyl group substituent on the aromatic ring, yielded stereoselective products when incubated for 6 hours with FMN and ammonium peroxydisulfate respectively, in the presence and absence of the dirigent protein. Incubations were carried out as described above with the following modifications: E-p-[9-³H]coumaryl (4 μmol ml⁻¹, 44.5 kBq) or E-[8-¹⁴C]sinapyl alcohols (4 μml⁻¹, 8.3 kBq) were used as substrates and, after 6 hour incubation at 30° C., the reaction mixture was extracted with EtOAc but without addition of radiochemical carriers. E-Sinapyl alcohol readily underwent coupling to afford syringaresinol, but chiral HPLC analysis revealed that the resulting products were, in every instance, racemic (Table 2). Interestingly, by itself, the 78-kD dirigent protein preparation catalyzed a low level of dimer formation, as previously noted, but only gave rise to racemic (±)-syringaresinol formation, which is presumably a consequence of the residual traces of contaminating oxidizing capacity present in the protein preparation.

In an analogous manner, no stereoselective coupling was observed with E-p-coumaryl alcohol as substrate. That is, only E-coniferyl alcohol undergoes stereoselective coupling in the presence of the dirigent protein. Given the marked substrate specificity of the dirigent protein for E-coniferyl alcohol, it will be of considerable interest to determine, in the future, how it differs from that affording (+)-syringaresinol in Eucommia ulmoides (T. Deyama, Chem. Pharm. Bull. 31, 2993 (1983)).

TABLE 2 Effect of dirigent protein on coupling of E-sinapyl alcohol (6 hour assay) E-Sinapyl alcohol in dimer equivalents Racemic Dirigent protein depleted (±)-syringaresinols (770 pmol ml⁻¹) (nmol ml⁻¹) (nmol ml⁻¹) FMN absent 570 ± 100 290 ± 40 (0.5 μmol ml⁻¹) present 610 ± 110 340 ± 40 Ammonium absent 1400 ± 120  1020 ± 40  peroxydisulfate present 1520 ± 10  1060 ± 30  (10 μmol ml⁻¹) Dirigent protein present 110 ± 10   50 ± 10

Although the inventors do not intend to be bound by any particular mechanism for stereoselective coupling, three distinct possibilities can be envisaged. The most likely is that the oxidase or oxidant generates free-radical species from E-coniferyl alcohol, and that the latter are the true substrates that bind to the dirigent protein prior to coupling. The other two possibilities would require that E-coniferyl alcohol molecules are bound and oriented on the dirigent protein, thereby ensuring that only (+)-pinoresinol formation occurs upon subsequent oxidative coupling: this could occur either if both substrate phenolic hydroxyl groups were exposed so that they could readily be oxidized by an oxidase or oxidant, or if an electron transfer mechanism were operative between the oxidase or oxidant and an electron acceptor site or sites on the dirigent protein.

Among the three alternative mechanisms, three lines of evidence suggest “capture” of phenoxy radical intermediates by the dirigent protein. First, the rates of both substrate depletion and product formation are largely unaffected by the presence of the dirigent protein. If capture of the free-radical intermediates is the operative mechanism, the dirigent protein would only affect the specificity of coupling when single-electron oxidation of coniferyl alcohol is rate-determining. Second, an electron transfer mechanism is currently ruled out, since we observed no new ultraviolet-visible chromophores in either the presence or absence of an auxiliary oxidase or oxidant, under oxidizing conditions. Third, preliminary kinetic data (as disclosed in Example 4) support the concept of free-radical capture based on the formal values of Michaelis constant (K_(m)) and maximum velocity (V_(max)) characterizing the conversion of E-coniferyl alcohol into (+)-pinoresinol, with the dirigent protein alone and in the presence of the various oxidases or oxidants.

EXAMPLE 4 Kinetic Characterization of the Conversion of E-Coniferyl Alcohol to (+)-pinoresinol in the Presence of Dirigent Protein and an Oxygenating Agent

Assays were carried out as described in Example 3 by incubating a series of E-[9-³H]coniferyl alcohol concentrations (between 8.00 and 0.13 μmol ml⁻¹, 7.3 MBq mole liter⁻¹) with dirigent protein (770 pmol ml⁻¹) alone and in presence of Forsythia laccase (2.1 pmol ml⁻¹), fraction III (12 μg protein ml⁻¹), or FMN (0.5 μmol ml⁻¹). Assays with dirigent protein, in presence or absence of FMN, were incubated at 30° C. for 1 hour, whereas assays with Forsythia laccase or fraction III in presence or absence of dirigent protein were incubated at 30° C. for 15 min. If free-radical capture by the dirigent protein is the operative mechanism, the Michaelis-Menten parameters obtained will only represent formal rather than true values, because the highest free-energy intermediate state during the conversion of E-coniferyl alcohol into (+)-pinoresinol is still unknown and the relation between the concentration of substrate and that of the corresponding intermediate free-radical in open solution has not been delineated.

Bearing these qualifications in mind, we estimated formal K_(m) and V_(max) values for the dirigent protein preparation. As noted earlier, it was capable of engendering formation of low levels of both (+)-pinoresinol from E-coniferyl alcohol, and racemic (±)-syringaresinols from E-sinapyl alcohol, because of traces of contaminating oxidizing capacity. With this preparation (Table 3), a formal K_(m) of 10±6 mM and V_(max) of 0.02±0.02 mol s⁻¹ mol⁻¹ were obtained. However, with addition of fraction III, laccase, and FMN, the formal K_(m) values (mM) were reduced to 1.6±0.3, 0.100±0.003, and 0.10±0.01, respectively, whereas the V_(max) values were far less affected at these concentrations of auxiliary oxidase/oxidant.

Formal K_(m) and V_(max) values were calculated for the laccase and fraction III oxidase with respect to E-coniferyl alcohol conversion into the three racemic lignans. However, no direct comparisons can be made to the 78-kD protein, since the formal K_(m) values involve only the corresponding oxidases. For completeness, the K_(m) (mM) and V_(max) (mol s⁻¹ mol⁻¹ enzyme) were as follows: with respect to the laccase, 0.200±0.001 and 3.9±0.2 for (±)-erythro/threo guaiacylglycerol 8-O-4′-coniferyl alcohol ethers, 0.3000±0.0003 and 13.1±0.6 for (±)-dehydrodiconiferyl alcohols, and 0.300±0.002 and 7.54±0.50 for (±)-pinoresinols; with respect to the fraction III oxidase (estimated to have a native molecular weight of 80 kDa), 2.2±0.3 and 0.20±0.03 for (±)-erythro/threo guaiacylglycerol 8-O-4′-coniferyl alcohol ethers, 2.2±0.2 and 0.7±0.1 for (±)-dehydrodiconiferyl alcohols, and 3.7±0.7 and 0.6±0.1 for (±)-pinoresinols.

These preliminary kinetic parameters are in harmony with the finding that dirigent protein does not substantially affect the rate of E-coniferyl alcohol depletion in the presence of fraction III, laccase and FMN. Both sets of results are together in accord with the working hypothesis that the dirigent protein functions by capturing free-radical intermediates which then undergo stereoselective coupling.

TABLE 3 Effect of various oxidants on formal K_(m) and V_(max) values for the dirigent protein (770 pmol ml⁻¹) during (+)-pinoresinol formation from E-coniferyl alcohol V_(max) (mol s⁻¹ mol⁻¹ Oxidase/Oxidant Formal K_(m) (mM) dirigent protein) Dirigent protein 10 ± 6  0.02 ± 0.02 Fraction III (12 μg protein ml⁻¹) 1.6 ± 0.3 0.10 ± 0.03 Laccase (2.07 pmol ml⁻¹) 0.100 ± 0.003 0.0600 ± 0.0002 FMN (0.5 μmol ml⁻¹) 0.10 ± 0.01 0.024 ± 0.001

EXAMPLE 5 Cloning of the Dirigent Protein cDNA from Forsythia intermedia

Plant Materials—Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, Minn.), and maintained in Washington State University greenhouse facilities, or were gifts from the local community.

Materials—All solvents and chemicals used were reagent or HPLC grade. Taq thermostable DNA polymerase was obtained from Promega, whereas restriction enzymes were from Gibco BRL (HaeIII), Boehringer Mannheim (Sau3a) and Promega (TaqI). pT7Blue T-vector and competent NovaBlue cells were purchased from Novagen and radiolabeled nucleotide ([α⁻³²P]dCTP) was from DuPont NEN.

Oligonucleotide primers for polymerase chain reaction (PCR) and sequencing were synthesized by Gibco BRL Life Technologies. GENECLEAN II® kits (BIO 101 Inc.) were used for purification of PCR fragments, with the gel-purified DNA concentrations determined by comparison to a low DNA mass ladder (Gibco BRL) in 1.5% agarose gels.

Instrumentation—UV (including RNA and DNA determinations at OD₂₆₀) spectra were recorded on a Lambda 6 UV/VIS spectrophotometer. A Temptronic II thermocycler (Thermolyne) was used for all PCR amplifications. Purification of DNA for sequencing employed a QIAwell Plus plasmid purification system (QIAGEN) followed by PEG precipitation (Sambrook, J., Fritsch, E. F., and Maniatis, T. (1994) Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.), with DNA sequences determined using an Applied Biosystems Model 373A automated sequencer. Amino acid sequences were obtained using an Applied Biosystems protein sequencer with on-line HPLC detection, according to the manufacturer's instructions.

Dirigent Protein Amino Acid Sequencing—The dirigent protein N-terminal amino acid sequence (SEQ ID NO:1) was obtained from the purified protein using an Applied Biosystems protein sequencer with on-line HPLC detection. For trypsin digestion, the purified enzyme (150 pmol) was suspended in 0.1 M Tris-HCl (50 μl, pH 8.5, Boehringer Mannheim, sequencing grade), with urea added to give a final concentration of 8 M in 77.5 μl. The mixture was incubated for 15 min at 50° C., following which 100 mM iodoacetamide (2.5 μl) was added, with the whole kept at room temperature for 15 min. Trypsin (1 μg in 20 μl) was then added, with the mixture digested for 24 h at 37° C., following which TFA (4 μl) was added to stop the enzymatic reaction. The resulting mixture was subjected to reversed phase HPLC analysis (C-8 column, Applied Biosytems), this being eluted with a linear gradient over 2 h from 0 to 100% acetonitrile (in 0.1% TFA) at a flow rate of 0.2 ml/min with detection at 280 nm. Fractions containing individual oligopeptide peaks were collected manually and directly submitted to amino acid sequencing (SEQ ID Nos:2-7).

Forsythia intermedia stem cDNA Library Synthesis—Total RNA (˜300 μg/g fresh weight) was obtained (Dong, Z. D., and Dunstan, D. I. (1996) Plant Cell Reports 15:516-521) from young green stems of greenhouse-grown Forsythia intermedia plants (var. Lynwood Gold). A Forsythia intermedia stem cDNA library was constructed using 5 μg of purified poly A⁺ mRNA (Oligotex-d™ Suspension, QIAGEN) with the ZAP-cDNA® synthesis kit, the Uni-ZAP™ XR vector and the Gigapack® II Gold packaging extract (Stratagene), with a titer of 1.2×10⁶ PFU for the primary library. A portion (30 ml) of the amplified library (1.2×10¹⁰ PFU/ml; 158 ml total) (Sambrook, J. et al., supra) was used to obtain pure cDNA library DNA (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidnam, J. G., Smith, J. A., and Struhl, K. (1991) Current Protocols in Molecular Biology, 2 volumes, Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, N.Y.) for PCR.

Dirigent Protein DNA Probe Synthesis—The N-terminal and internal peptide amino acid sequences were used to construct the degenerate oligonucleotide primers. Purified F. intermedia cDNA library DNA (5 ng) was used as the template in 100 μl PCR reactions (10 mM Tris-HCl [pH 9.0], 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl₂, 0.2 mM each dNTP and 2.5 units Taq DNA polymerase) with primer PSINT1 (SEQ ID NO:8) (100 pmol) and either primer PSI7R (SEQ ID NO:11) (20 pmol), primer PSI2R (SEQ ID NO:10) (20 pmol) or primer PSI1R (SEQ ID NO:9) (20 pmol). PCR amplification was carried out in a thermocycler as follows: 35 cycles of 1 min at 94° C., 2 min at 50° C. and 3 min at 72° C.; with 5 min at 72° C. and an indefinite hold at 4° C. after the final cycle. Single-primer, template-only and primer-only reactions were performed as controls. PCR products were resolved in 1.5% agarose gels, where a single band (˜370-, ˜155- or ˜125-bp, respectively) was observed for each reaction.

To determine the nucleotide sequence of the amplified bands, five 100 μl PCR reactions were performed as above with PSINT1 (SEQ ID NO:8) +PSI7R (SEQ ID NO:11), PSINT1 (SEQ ID NO:8) +PSI2R (SEQ ID NO:10) and PSINT1 (SEQ ID NO:8) +PSI1R (SEQ ID NO:9) primer pairs. The 5 reactions from each primer pair were concentrated (Microcon 30, Amicon Inc.) and washed with TE buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA; 2×200 μl), with the PCR products subsequently recovered in TE buffer (2×50 μl). These were resolved in preparative 1.5% agarose gels. Each gel-purified PCR product (˜0.2 pmol) was then ligated into the pT7Blue T-vector and transformed into competent NovaBlue cells, according to Novagen's instructions. Insert sizes were determined using the rapid boiling lysis and PCR technique (with R20mer and U19mer primers) according to the manufacturer's instructions. Restriction analyses were performed to determine whether all inserts from the reactions utilizing each of the foregoing primer pairs were the same, as follows: to 20 μl each of a 100 μl PCR reaction (insert of interest amplified with R20mer(SEQ ID NO:74) and U19mer(SEQ ID NO:75) primers) were added 4 units HaeIII, 1.5 units Sau3a or 5 units TaqI restriction enzyme. Restriction digestions were allowed to proceed for 60 min at 37° C. for HaeIII and Sau3A and at 65° C. for TaqI reactions. Restriction products were resolved in 1.5% agarose gels giving one restriction group for each insert tested. Five recombinant plasmids from PSINT1 (SEQ ID NO:8) +PSI7R (SEQ ID NO:11) (called pT7PSI1-pT7PSI5) and 2 recombinant plasmids from PSINT1 (SEQ ID NO:8) +PSI2R (SEQ ID NO:10) (called pT7PSI6 and pT7PSI7) PCR products were selected for DNA sequencing; all contained the same open reading frame (ORF) (SEQ ID NO:69). The dirigent protein probe was next constructed as follows: five 100 μl PCR reactions were performed as above with 10 ng pT7PSI1 DNA (SEQ ID NO:69) with primers PSINT1 (SEQ ID NO:8) and PSI7R (SEQ ID NO:11). Gel-purified pT7PSI1 insert (50 ng) was used with Pharmacia's ^(T7)QuickPrime® kit and [α-³²P]dCTP, according to kit instructions, to produce a radiolabeled probe (in 0.1 ml), which was purified over BioSpin 6 columns (Bio-Rad) and added to carrier DNA (0.5 mg/ml sheared salmon sperm DNA [Sigma], 0.9 ml).

Library Screening—600,000 PFU of F. intermedia amplified cDNA library were plated for primary screening, according to Stratagene's instructions. Plaques were blotted onto Magna Nylon membrane circles (Micron Separations Inc.), which were then allowed to air dry. The membranes were placed between two layers of Whatman® 3MM Chr paper. cDNA library phage DNA was fixed to the membranes and denatured in one step by autoclaving for 2 min at 100° C. with fast exhaust. The membranes were washed for 30 min at 37° C. in 6×standard saline citrate (SSC) and 0.1% SDS and prehybridized for 5 h with gentle shaking at 57-58° C. in preheated 6×SSC, 0.5% SDS and 5×Denhardt's reagent (hybridization solution, 300 ml) in a crystallization dish (190×75 mm). The [³²P]radiolabeled probe was denatured (boiling, 10 min), quickly cooled (ice, 15 min) and added to a preheated fresh hybridization solution (60 ml, 58° C.) in a crystallization dish (150×75 mm). The prehybridized membranes were next added to this dish, which was then covered with plastic wrap. Hybridization was performed for 18 h at 57-58° C. with gentle shaking. The membranes were washed in 4×SSC and 0.5% SDS for 5 min at room temperature, transferred to 2×SSC and 0.5% SDS (at room temperature) and incubated at 57-58° C. for 20 min with gentle shaking, wrapped with plastic wrap to prevent drying and finally exposed to Kodak X-OMAT AR film for 24 h at −80° C. with intensifying screens. Twenty positive plaques were purified through two more rounds of screening with hybridization conditions as above.

In vivo Excision and Sequencing of Dirigent Protein cDNA-containing Phagemids—Purified cDNA clones were rescued from the phage following Stratagene's in vivo excision protocol. Both strands of several different cDNAs that coded for dirigent protein were completely sequenced using overlapping sequencing primers. Two distinct cDNAs were identified, called pPSD_Fi1(SEQ ID NO:12) and pPSD_Fi2(SEQ ID NO:14).

Sequence Analysis—DNA and amino acid sequence analyses were performed using the Unix-based GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711; Rice, P. (1996) Program Manual for the EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1Rq, England) and the ExPASy World Wide Web molecular biology server (Geneva University Hospital and University of Geneva, Geneva, Switzerland).

EXAMPLE 6 Expression of Functional Dirigent Protein in Spodoptera frugiperda

Attempts to express functional dirigent protein in Escherichia coli failed. Consequently, we expressed the dirigent protein in Spodoptera frugiperda utilizing a baculovirus expression system. The full-length 1.2 kb cDNA clone for the dirigent protein (PSD) in F. intermedia, containing both the 5′ and 3′ untranslated regions, was excised from the pBlueScript (Stratagene) derived plasmid pPSD_Fi1 (SEQ ID NO:12) using the restriction endonucleases BamH I and Xho I. This 1.2 kb fragment was directionally subcloned into these same restriction sites in the multiple cloning site of the baculovirus transfer vector pBlueBac4 (Invitrogen, San Diego, Calif.). This produced the 6.0 kb construct pBB4/PSD which generates a non-fusion dirigent protein with translation being initiated at the dirigent protein cDNA start codon. This construct was then co-transfected with linearized Bac-N-Blue DNA (Invitrogen) into Spodoptera frugiperda Sf9 cells by the technique of cationic liposome mediated transfection to produce, by means of homologous recombination, the recombinant Autographa californica nuclear polyhedrosis viral (AcMNPV) DNA Bac-N-Blue dirigent protein (BB/PSD) which was purified from plaques according to procedures described by Invitrogen. The final recombinant AcMNPV-BB/PSD contains the PSD gene under the polyhedrin promoter control and the essential sequence needed for replication of the recombinant virus. To verify that the dirigent protein was successfully expressed in the insect cell culture, log phase Sf9 cells infected with the AcMNPV-PSD recombinant viral high titer stock were used to obtain heterologous protein production. Maximal dirigent protein yield occurred by 48-70 hours post-infection. As determined by SDS-PAGE and (+)-pinoresinol forming activity, the protein was found secreted into the medium and showed a molecular mass and activity which corresponded to the indigenous protein originally isolated from Forsythia intermedia.

EXAMPLE 7 Isolation of Dirigent Protein Clones from Thuja plicata and Tsuga heterophylla

The coding region of a Forsythia dirigent protein cDNA, psd-Fi1 (SEQ ID NO:12), was used to screen cDNA libraries from Thuja plicata and Tsuga heterophylla. The conditions and methods were as disclosed in Example 5, except that hybridization was carried out at 45-50° C. Two dirigent protein cDNAs were isolated from Tsuga heterophylla (SEQ ID Nos:16, 18), and eight dirigent protein cDNAs were isolated from Thuja plicata (SEQ ID Nos:20, 22, 24, 26, 28, 30, 32, 34).

EXAMPLE 8 Purification of Pinoresinol/lariciresinol Reductases from Forsythia Intermedia

Plant Materials. Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, Minn.), and maintained in Washington State University greenhouse facilities, or were gifts from the local community.

Materials. All solvents and chemicals used were reagent or HPLC grade. Unlabeled (±)-pinoresinols and (±)-lariciresinols were synthesized as described (Katayama, T. et al., Phytochemistry 32:581-591 (1993)). [4R-3H]NADPH was obtained as previously reported (Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)) by modification of the procedure of Moran et al. (Moran, R. G. et al., Anal. Biochem. 138:196-204 (1984)), and [4R-2H]NADPH was prepared according to Anderson and Lin (Anderson, J. A., and Lin B. K., Phytochemistry 32:811-812 (1993)). Yeast glucose-6-phosphate dehydrogenase (Type IX,22.32. mmol h⁻¹ mg⁻¹) and yeast hexokinase (Type F300, 15.12 mmol⁻¹ mg⁻¹) were purchased from Sigma and dihydrofolate reductase (Lactobacillus casei, 33.48 mmol h⁻¹ mg⁻¹) was obtained from Biopure Co. Affi-Gel Blue Gel (100-200 mesh) and Bio-Gel HT Hydroxyapatite were purchased from Bio-Rad, whereas Phenyl Sepharose CL-4B, MonoQ HR 5/5, MonoP HR 5/20, Superose 6, Superose 12, Superdex 75, PD-10 columns, molecular weight standards and Polybuffer 74 were obtained from Pharmacia LKB Biotechnology, Inc. Adenosine 2′,5′-diphosphate Sepharose and Reactive Yellow 3 Agarose were from Sigma Chemical Co.

Instrumentation. ¹H Nuclear magnetic resonance spectra (300 and 500 MHz) were recorded on Brüker AMX300 and Varian VXR500S spectrometers, respectively, using CDCl₃ as solvent with chemical shifts (δ ppm) reported downfield from tetramethylsilane (internal standard). UV (including RNA and DNA determinations at OD₂₆₀) and mass spectra were obtained on Lambda 6 UV/VIS and VG 7070E (ionizing voltage 70 eV) spectrophotometers, respectively. High performance liquid chromatography was carried out using either reversed-phase (Waters, Nova-pak C 18, 150×3.9 mm inner diameter) or chiral (Daicel, Chiralcel OD or Chiralcel OC, 240×4.6 mm inner diameter) columns, with detection at 280 nm (Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)). Radioactive samples were analyzed in Ecolume (ICN) and measured using a liquid scintillation counter (Packard, Tricarb 2000 CA). Amino acid sequences were obtained using an Applied Biosystems protein sequencer with on-line HPLC detection, according to the manufacturer's instructions.

Enzyme Assays. Pinoresinol and lariciresinol reductase activities were assayed by monitoring the formation of (+)-[³H]lariciresinol and (−)-[³H]secoisolariciresinol (Chu, A. et al., J. Biol. Chem. 268:27026-27033 (1993)).

Briefly, each assay for pinoresinol reductase activity consisted of (±)-pinoresinols (5 mM in MeOH, 20 μl), the enzyme preparation at the corresponding stage of purity (100 μl), and buffer (20 mM Tris-HCl, pH 8.0, 110 μl). The enzymatic reaction was initiated by addition of [4R-³H]NADPH (10 mM, 6.79 kBq/mmol in 20 μl of double-distilled H₂O). After 30 min incubation at 30° C. with shaking, the assay mixture was extracted with EtOAc (500 μl) containing (±)-lariciresinols (20 μg) and (±)-secoisolariciresinols (20 μg) as radiochemical carriers. After centrifugation (13,800×g, 5 min), the EtOAc solubles were removed and the extraction procedure was repeated. For each assay, the EtOAc solubles were combined with an aliquot (100 μl) removed for determination of its radioactivity using liquid scintillation counting. The remainder of the combined EtOAc solubles was evaporated to dryness in vacuo, reconstituted in MeOH/3% acetic acid in H₂O (30:70, 100 μl) and subjected to reversed phase and chiral column HPLC. Controls were performed using either denatured enzyme (boiled for 10 min) or in the absence of (±)-pinoresinols as substrate.

Lariciresinol reductase activity was assayed by monitoring the formation of (−)-[³H]secoisolariciresinol. These assays were carried out exactly as described above, except that (±)-lariciresinols (5 mM in MeOH, 20 μl) were used as substrates, with (±)-secoisolariciresinols (20 μg) added as radiochemical carriers.

General Procedures for Enzyme Purification. Protein purification procedures were carried out at 4° C. with chromatographic eluents monitored at 280 nm, unless otherwise stated. Protein concentrations were determined by the method of Bradford (Bradford, M. M., Anal. Biochem. 72:248-254 (1976)) using γ-globulin as standard. Polyacrylamide gel electrophoresis used gradient (4-15%, Bio-Rad) gels under denaturing and reducing conditions, these being performed in Laemmli's buffer system (Laenimli, U. K., Nature 227:680-685 (1970)). Proteins were visualized by silver staining (Morrissey, J. H., Anal. Biochem. 117:307-310 (1981)).

Preparation of crude extracts. F. intermedia stems (20 kg) were harvested, cut into 3-6 cm sections, and stored at −20° C. until needed. Batches of stems (2 kg) were frozen in liquid nitrogen and pulverized in a Waring Blendor. The resulting powder was homogenized with potassium phosphate buffer (0.1 mM, pH 7.0, 4 L), containing 5 mM dithiothreitol. The homogenate was filtered through four layers of cheesecloth into a beaker containing 10% (w/v) polyvinylpolypyrolidone. The filtrate was centrifuged (12,000×g, 15 min). The resulting supernatant was fractionated with (NH₄)₂SO₄, with proteins precipitating between 40 and 60% saturation recovered by centrifugation (10,000×g, 1 h). The pellet was next reconstituted in a minimum amount of Tris-HCl buffer (20 mM, pH 8.0), containing 5 mM dithiothreitol (buffer A) and desalted using prepacked PD-10 columns (Sephadex G-25 medium) equilibrated with buffer A.

Affinity (Affi Blue Gel) Chromatography. The crude enzyme preparation (191 mg in buffer A, 5 nmol h⁻¹ mg⁻¹) was applied to an Affi Blue Gel column (2.6×70 cm) equilibrated in buffer A. After washing the column with 200 ml of buffer A, pinoresinol/lariciresinol reductase was eluted with a linear NaCl gradient (1.5-5 M in 300 ml) in buffer A at a flow rate of 1 ml min⁻¹. Active fractions were stored (−80° C.) until needed.

Hydrophobic Interaction Chromatography (Phenyl Sepharose). After thawing, ten preparations resulting from the Affi Blue chromatography step (150 mg, 51 nmol h⁻¹ mg⁻¹) were combined and applied to a Phenyl Sepharose column (1×10 cm) equilibrated in buffer A, containing 5 M NaCl. The column was washed with two bed volumes of the same buffer. Pinoresinol/lariciresinol reductase was eluted using a linear gradient of decreasing concentration of NaCl (5-0 M in 40 ml) in buffer A at a flow rate of 1 ml min⁻¹. Fractions catalyzing pinoresinol/lariciresinol reduction were combined and pooled.

Hydroxyapatite I Chromatography. Active protein (31 mg, 91 nmol h⁻¹ mg⁻¹) from the phenyl sepharose purification step was applied to an hydroxyapatite column (1.6×70 cm) equilibrated in 10 mM potassium phosphate buffer, pH 7.0, containing 5 mM dithiothreitol (buffer B). Pinoresinol/lariciresinol reductase was eluted with a linear gradient of potassium phosphate buffer, pH 7.0 (0.01-0.4 M in 200 ml) at a flow rate of 1 ml min⁻¹. Active fractions were combined. The buffer was then exchanged with buffer A using PD-10 prepacked columns.

Affinity (2′,5′-ADP Sepharose) Chromatography. The enzyme solution resulting from the hydroxyapatite purification step (6.5 mg, 463 nmol h⁻¹ mg⁻¹) was next loaded on a 2′,5′-ADP Sepharose (1×10 cm) column, previously equilibrated in buffer A containing 2.5 mM EDTA (buffer A′) and then washed with 25 ml of buffer A′. Pinoresinol/lariciresinol reductase was eluted with a step gradient of NADP+ (0.3 mM in 10 ml) in buffer A′ at a flow rate of 0.5 ml min⁻¹. [NAD+ (up to 3 mM) did not elute pinoresinol/lariciresinol reductase activity.] Because of the interference of the absorbance of the NADP+, it was not possible to directly monitor the eluent at 280 nm. Protein concentrations for each fraction were determined spectrophotometrically according to Bradford (Bradford, M. M., Anal. Biochem. 72:248-254 (1976)).

Hydroxyapatite II Chromatography. Fractions from the 2′,5′-ADP Sepharose column that exhibited pinoresinol/lariciresinol reductase activity (0.85 mg, 1051 nmol h⁻¹ mg⁻¹) were combined and directly applied to a second hydroxyapatite column (1×3 cm), equilibrated in buffer B, with the enzyme eluted with a linear gradient of potassium phosphate buffer, pH 7.0 (0.01-0.4 M in 45 ml) at a flow rate of 1 ml min⁻¹.

Affinity (Affi Yellow) Chromatography—Active fractions (160 μg, 7960 nmol h⁻¹ mg⁻¹) from the second hydroxyapatite column purification step were next applied to a Reactive Yellow 3 Agarose column (1×3 cm), equilibrated in buffer A. Pinoresinol/lariciresinol reductase was eluted with a linear NaCl gradient (0-2.5 M in 100 ml) at a flow rate of 1 ml min⁻¹.

Fast Protein Liquid Chromatography (Superose 12 Chromatography)—Combined fractions from the Affi Yellow purification step having the highest activity (50 μg, 10,940 nmol h⁻¹ mg⁻¹) were pooled and concentrated to 1 ml, using a Centricon 10 microconcentrator (Amicon, Inc.). The enzyme solution was then applied in portions of 200 μl to a fast protein liquid chromatography column (Superose 12, HR 10/30). Gel filtration was performed in a buffer containing 20 mM Tris-HCl, pH 8.0, 150 mM NaCl and 5 mM dithiothreitol at a flow rate of 0.4 ml min⁻¹. Pinoresinol/lariciresinol reductase was eluted with 12.8 ml of the mobile phase. The active fractions which coincided with the UV profile (absorbance at 280 nm) were pooled (20 μg, 15,300 nmol h⁻¹ mg⁻¹) and desalted (PD-10 prepacked columns).

The foregoing purification protocol resulted in a 3060-fold purification of (+)-pinoresinol/(+)-lariciresinol reductase. As for many of the enzymes involved in phenylpropanoid metabolism, the protein was in very low abundance, i.e. 20 kg F. intermedia stems yielded only ˜20 μg of the purified (+)-pinoresinol/(+)-lariciresinol reductase.

EXAMPLE 9 Characterization of Purified Pinoresinol/lariciresinol Reductases from Forsythia Intermedia

Isoelectric Focussing and pI Determination. In all stages of the purification protocol, (+)-pinoresinol/(+)-lariciresinol reductase activities coeluted. Given this observation, it was essential to unambiguously ascertain whether more than one form of the protein existed, i.e., whether one form of the protein catalyzed the reduction of pinoresinol, and another form of the protein catalyzed the reduction of lariciresinol. To this end, the isoelectric point of pinoresinol/lariciresinol reductase was estimated by chromatofocussing on a MonoP HR 5/20 FPLC column.

Active fractions from the Superose 12 gel filtration column (Example 1) were pooled and the buffer exchanged with 25 mM Bis-Tris, pH 7.1, using prepacked PD-10 columns, equilibrated in the same buffer. The preparation so obtained was loaded on the chromatofocussing column and a pH gradient between 7.1 and 3.9 was formed, using Polybuffer 74 as eluent at a flow rate of 0.5 ml min⁻¹. Aliquots (200 μl) of each fraction were assayed for pinoresinol/lariciresinol reductase activities. The remainder of the fractions was used to determine the pH gradient.

Molecular Weight Determination. Application of the MonoP HR 5/20 FPLC column preparation of pinoresinol/lariciresinol reductase to SDS-gradient gel electrophoresis (4-15% polyacrylamide) revealed the presence of two protein bands of similar apparent molecular weight, whose separation was achieved via anion-exchange chromatography on a MonoQ HR 5/5 FPLC matrix. Pooled fractions from the Sepharose 12 purification step (Example 1) were applied to a MonoQ HR 5/5 column (Pharmacia), equilibrated in buffer A. The column was washed with 10 ml of buffer A and pinoresinol/lariciresinol reductase activity eluted using a linear NaCl gradient (0-500 mM in 50 ml) in buffer A at a flow rate of 0.5 ml min⁻¹. Aliquots (30 μl) of the collected fractions were analyzed by SDS polyacrylamide gel electrophoresis, using a gradient (4-15% acrylamide) gel. Proteins were visualized by silver staining. Active fractions 34 through 37 (27,760 nmol h⁻¹ mg⁻¹) and 38 through 41 (30,790 nmol h⁻¹ mg⁻¹) were pooled separately and immediately used for characterization.

The two protein bands thus resolved under denaturing conditions had apparent molecular masses of ˜36 and ˜35 kDa, respectively. Each of the two reductase forms had a pI˜5.7.

Native molecular weights of each reductase isoform were estimated via comparison of their elution behavior on Superose 12, Superose 6 and Superdex 75 gel filtration FPLC columns with the elution behavior of calibrated molecular weight standards. Gel filtration was carried out as set forth in Example 8. For each reductase, an apparent native molecular weight of 59,000 was calculated based on its elution volume, in contrast to that of ˜36,000 and ˜35,000 by SDS-polyacrylamide gel electrophoresis. While the discrepancy between molecular weights from gel filtration and SDS-PAGE remains unknown, it can tentatively be proposed that although the native protein likely exists as a dimer, it could also be a monomer of asymmetric shape, thereby altering its effective Stokes radius (Cantor, C. R., and Shimmel, P. R., Biophysical Chemistry, Part II, W.H. Freeman and Company, San Francisco, Calif. (1980); Stellwagen, E., Methods in Enzymology 182:317-328 (1990)), as reported for human thioredoxin reductase (Oblong, J. E., et al., Biochemistry 32:7271-7277 (1993)) and yeast metalloendopeptidase (Hrycyna, C. A., and Clarke, S., Biochemistry 32:11293-11301 (1993)).

pH and Temperature Optima. To determine the pH-optimum of pinoresinol/lariciresinol reductase, the enzyme preparation from the gel Superose 12 filtration step (Example 8) was assayed utilizing standard assay conditions (Example 8), except that the buffer was replaced with 50 mM Bis-Tris Propane buffer in the pH range of 6.3 to 9.4. The pH optimum was found to be pH 7.4.

The temperature optimum of pinoresinol/lariciresinol reductase was examined in the range between 4° C. and 80° C. under standard assay conditions (Example 8) utilizing the enzyme preparation from the gel filtration step (Example 8). At optimum pH, the temperature optimum for the reductase activity was established to be ˜30° C.

Kinetic Parameters. Velocity studies were carried out to ascertain whether the two reductase isoforms catalyzed distinct reductions, i.e., that of the conversion of (+)-pinoresinol to (+)-lariciresinol, and (+)-lariciresinol to (−)-secoisolariciresinol, respectively, or whether either displayed a preference for (+)-pinoresinol or (+)-lariciresinol as substrates. The initial velocity studies were carried out individually utilizing the two isoforms of the enzyme, and individually employing both (+)-pinoresinol and (+)-lariciresinol as substrates. Initial velocity studies were performed in triplicate experiments, using 50 mM Bis-Tris Propane buffer, pH 7.4 containing 5 mM dithiothreitol, pure enzyme (after MonoQ anion-exchange chromatography), ten different substrate concentrations (between 8.8 and 160 μM) at a constant NADPH concentration (80 μM). Incubations were carried out at 30° C. for 10 min (within the linear kinetic range). Kinetic parameters were determined from Lineweaver-Burk plots.

Importantly, the kinetic parameters were essentially the same for both the 35 kDa and the 36 kDa forms of the enzyme (i.e., Km for pinoresinol: 27±1.5 μm for the 35 kDa form of the enzyme, and 23±1.3 μM for the 36 kDa form of the enzyme; Km for lariciresinol: 121±5.0 μM for the 35 kDa form of the enzyme and 123±6.0 μM for the 36 kDa form of the enzyme). In an analogous manner, apparent maximum velocities (expressed as μmol h⁻¹ mg⁻¹ of protein) were also essentially identical (i.e., Vmax for pinoresinol: 16.2±0.4 for the 35 kDa form of the enzyme and 17.3±0.5 for the 36 kDa form of the enzyme; for lariciresinol: 25.2±0.7 for the 35 kDa form of the enzyme and 29.9±0.7 for the 36 kDa form of the enzyme). Thus, all available evidence suggests that (+)-pinoresinol/(+)-lariciresinol reductase exists as two isoforms, with each capable of catalyzing the reduction of both substrates. How this reduction is carried out, i.e., whether both reductions are done in tandem, in either quinone or furano ring form, awaits further study using a more abundant protein source.

Enzymatic Formation of (+)-[7′R-²H]Lariciresinol. Since the two (+)-pinoresinol/(+)-lariciresinol reductase isoforms exhibited essentially identical catalytic characteristics, the Sepharose 12 enzyme preparation (Example 8), containing both isoforms, was used to examine the stereospecificity of the hydride transfer. The strategy adopted utilized selective deuterium labeling using NADP²H as cofactor for the reduction of (+)-pinoresinol, with the enzymatic product, (+)-lariciresinol, being analyzed by ¹H NMR and mass spectroscopy. Thus, a solution of (±)-pinoresinols (5.2 mM in MeOH, 4 ml) was added to Tris-HCl buffer (20 mM, pH 8.0, containing 5 mM dithiothreitol, 22 ml) and stereospecifically deutero-labeled [4R-²H]NADPH (20 mM in H₂O, 4 ml) prepared via the method of Anderson and Lin (Anderson, J. A., and Lin B. K., Phytochemistry 32:811-812 (1993)), with the whole added to the enzyme preparation (20 ml). After incubation at 30° C. for 1 h with shaking, the assay mixture was extracted with EtOAc (2×50 ml). The EtOAc soluble fraction was combined, washed with saturated NaCl (50 ml), dried (Na₂SO₄), and evaporated to dryness in vacuo. The resulting extract was reconstituted in a minimum amount of EtOAc, applied to a silica gel column (0.5×7 cm), and eluted with EtOAc/hexanes (1:2). Fractions containing the enzymatic product were combined and evaporated to dryness.

The enzymatic product was established to be (+)-[7′R-²H]lariciresinol, as evidenced by the disappearance of the 7′-proR proton at δ 2.51 ppm due to its replacement by deuterium and by its molecular ion at (m/z) 361 (M++1) corresponding to the presence of one deuterium atom at C-7. ¹H NMR (300 MHz) (CDCl₃): 2.39 (m,¹H, C8H), 2.71 (m,¹H, C8′H), 2.88 (δ,¹H, J7′S,8′=5.0 Hz, C7′HS), 3.73 (δδ,¹H, J8′,9′b=7.0 Hz, J9′a,9′b=8.5 Hz, C9′Hβ), 3.76 (δδ,¹H, J8,9S=6.5 Hz, J9R,9S=8.5 Hz, C9HS), 3.86 (s,³H, OCH₃), 3.88 (s,³H, OCH₃), 3.92 (δδ,¹H, J8,9R=6.0 Hz, J9R,9S=9.5 Hz, C9HR), 4.04 (δδ,¹H, J8′,9′a=7.0 Hz, J9′a9′b=8.5 Hz, C9′Ha), 4.77 (δ,¹H, J7,8=6.6 Hz, C7H), 6.68-6.70 (m,²H, ArH), 6.75-6.85 (m,4H, ArH); MS m/z (%): 361 (M++1, 71.2), 360 (M+, 31.1), 237 (11.1), 153 (41.5), 152 (20.2), 151 (67.0), 138 (100), 137 (71.1).

Thus, hydride transfer from (+)-pinoresinol to (+)-lariciresinol had occurred in a manner whereby only the 7′-proR hydrogen position of (+)-lariciresinol was deuterated. An analogous result was observed for the conversion of (+)-lariciresinol into (−)-secoisolariciresinol, thereby establishing that the overall hydride transfer was completely stereospecific.

EXAMPLE 10

Amino Acid Sequence Analysis of Purified Pinoresinol/Lariciresinol Reductase from Forsythia intermedia

Pinoresinol/Lariciresinol Reductase Amino Acid Sequencing. The (+)-pinoresinol/(+)-lariciresinol reductase N-terminal amino acid sequence was obtained from each of the purified proteins, and a mixture of both, using an Applied Biosystems protein sequencer with on-line HPLC detection. The N-terminal sequence was the same for both isoforms (SEQ ID NO:36).

For trypsin digestion, 150 pmol of the enzyme purified from the Sepharose 12 column (Example 8) was suspended in 0.1 M Tris-HCl (50 μl, pH 8.5), with urea added to give a final concentration of 8 M in 77.5 μl. The mixture was incubated for 15 min at 50° C., then 100 mM iodoacetamide (2.5 μl) was added, with the whole kept at room temperature for 15 min. Trypsin (1 μg in 20 μl) was then added, with the mixture digested for 24 h at 37° C., after which TFA (4 μl) was added to stop the enzymatic reaction.

The resulting mixture was subjected to reversed phase HPLC analysis (C-8 column, Applied Biosytems), this being eluted with a linear gradient over 2 h from 0 to 100% acetonitrile (in 0.1% TFA) at a flow rate of 0.2 ml/min with detection at 280 nm. Fractions containing individual oligopeptide peaks were collected manually and directly submitted to amino acid sequencing. Four tryptic fragments were resolved in sufficient quantity to permit amino acid sequence determination. (SEQ ID Nos:37-40).

Cyanogen bromide digestion was performed by incubation of 150 pmol of the reductase purified from the Sepharose 12 column (Example 8) with 0.5 M cyanogen bromide in 70% formic acid for 40 h at 37° C., following which the cyanogen bromide and formic acid were removed by centrifugation under reduced pressure (SpeedVac). The resulting oligopeptide fragments were separated by HPLC and three were resolved in sufficient quantity to permit sequencing (SEQ ID Nos:41-43).

EXAMPLE 11 Cloning of Pinoresinol/Lariciresinol Reductase from Forsythia intermedia

Plant Materials. Forsythia intermedia plants were either obtained from Bailey's Nursery (var. Lynwood Gold, St., Paul, Minn.), and maintained in Washington State University greenhouse facilities, or were gifts from the local community.

Materials. All solvents and chemicals used were reagent or HPLC grade. UV RNA and DNA determinations at OD₂₆₀ were obtained on a Lambda 6 UV/VIS spectrophotometer. A Temptronic II thermocycler (Thermolyne) was used for all PCR amplifications. Taq thermostable DNA polymerase was obtained from Promega, whereas restriction enzymes were from Gibco BRL (HaeIII), Boehringer Mannheim (Sau3a) and Promega (TaqI). pT7Blue T-vector and competent NovaBlue cells were purchased from Novagen and radiolabeled nucleotides ([α-³²P]dCTP and [γ-³²P]ATP) were from DuPont NEN.

Oligonucleotide primers for polymerase chain reaction (PCR) and sequencing were synthesized by Gibco BRL Life Technologies. GENECLEAN II® kits (BIO 101 Inc.) were used for purification of PCR fragments, with the gel-purified DNA concentrations determined by comparison to a low DNA mass ladder (Gibco BRL) in 1.5% agarose gels.

Forsythia RNA Isolation. Initial attempts to isolate functional F. intermedia RNA from fast-growing, green stem tissue were unsuccessful, due to difficulties encountered via facile oxidation by its plant phenolic constituents. This problem was, however, successfully overcome by utilization of an RNA isolation procedure, specifically designed for woody plant tissue, which uses low pH and reducing conditions in the extraction buffer to prevent oxidation (Dong, Z. D., and Dunstan, D. I., Plant Cell Reports 15: 516-521(1996)).

Forsythia intermedia stem cDNA Library Synthesis. Total RNA (˜300 μg/g fresh weight) was obtained from young green stems of greenhouse-grown Forsythia intermedia plants (var. Lynwood Gold) (Dong, Z. D., and Dunstan, D. I., Plant Cell Reports 15:516-521 (1996)). A Forsythia intermedia stem cDNA library was constructed using 5 μg of purified poly A+mRNA (Oligotex-dT™ Suspension, QIAGEN) with the ZAP-cDNA® synthesis kit, the Uni-ZAP™ XR vector and the Gigapack® II Gold packaging extract (Stratagene), with a titer of 1.2×10⁶ PFU for the primary library. A portion (30 ml) of the amplified library (1.2×10¹⁰ PFU/ml; 158 ml total) was used to obtain pure cDNA library DNA for PCR (Sambrook, J. et al., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994); Ausubel, F. M. et al., Current Protocols in Molecular Biology, 2 volumes, Greene Publishing Associates and Wiley-Interscience, John Wiley & Sons, NY (1991)).

Pinoresinol/Lariciresinol Reductase DNA Probe Synthesis—The N-terminal and internal peptide amino acid sequences were used to construct the degenerate oligonucleotide primers. Specifically, the primer PLRN5 (SEQ ID NO:44) was based on the sequence of amino acids 7 to 13 of the N-terminal peptide (SEQ ID NO:36). The primer PLR14R (SEQ ID NO:45) was based on the sequence of amino acids 2 to 8 of the internal peptide sequence set forth in (SEQ ID NO:37). The primer PLR15R (SEQ ID NO:46) was based on the sequence of amino acids 9 to 15 of the internal peptide sequence set forth in (SEQ ID NO:37). The sequence of amino acids 9 to 15 of the internal peptide sequence set forth in SEQ ID NO:37, upon which the sequence of primer PLR15R (SEQ ID NO:46) was based, also corresponded to the sequence of amino acids 4 to 10 of the cyanogen bromide-generated, internal fragment set forth in SEQ ID NO:41.

Purified F. intermedia cDNA library DNA (5 ng) was used as the template in 100 μl PCR reactions (10 mM Tris-HCl [pH 9.0], 50 mM KCl, 0.1% Triton X-100, 2.5 mM MgCl₂, 0.2 mM each dNTP and 2.5 units Taq DNA polymerase) with primer PLRN5 (SEQ ID NO:44) (100 pmol) and either primer PLRI5R (SEQ ID NO:46) (20 pmol) or primer PLRI4R (SEQ ID NO:45) (20 pmol). PCR amplification was carried out in a thermocycler as follows: 35 cycles of 1 min at 94° C., 2 min at 50° C. and 3 min at 72° C.; with 5 min at 72° C. and an indefinite hold at 4° C. after the final cycle. Single-primer, template-only and primer-only reactions were performed as controls. PCR products were resolved in 1.5% agarose gels. The combination of primers PLRN5 (SEQ ID NO:44) and PLRI4R (SEQ ID NO:45) yielded a single band of 380-bp corresponding to bases 22 to 393 of SEQ ID NO:47. The combination of primers PLRN5 (SEQ ID NO:44) and PLRI5R (SEQ ID NO:46) yielded a single band of 400-bp corresponding to bases 22 to 423 of SEQ ID NO:47.

To determine the nucleotide sequence of the two amplified bands, five, 100 μl PCR reactions were performed as above with each of the following combinations of template and primers: 380 bp amplified product plus primers PLRN5 (SEQ ID NO:44) and PLRI4R (SEQ ID NO:45); 400 bp amplified product plus primers PLRN5 (SEQ ID NO:44) and PLRI5R (SEQ ID NO:46). The 5 reactions from each combination of primers and template were concentrated (Microcon 30, Amicon Inc.) and washed with TE buffer (10 mM Tris-HCI, pH 8.0, 1 mM EDTA; 2×200 μl), with the PCR products subsequently recovered in TE buffer (2×50 μl). These were resolved in preparative 1.5% agarose gels. Each gel-purified PCR product (˜0.2 pmol) was then ligated into the pT7Blue T-vector and transformed into competent NovaBlue cells, according to Novagen's instructions. Insert sizes were determined using the rapid boiling lysis and PCR technique (utilizing R20mer (SEQ ID NO:74) and U19mer (SEQ ID NO:75) primers according to the manufacturer's (Novagen's) instructions.

Restriction analysis was performed to determine whether all inserts for each combination of primers and template were the same. Restriction analysis was carried out as follows: each of the inserts was amplified by PCR utilizing the R20 (SEQ ID NO:74) and U19 (SEQ ID NO:75) primers. To 20 μl each of a 100 μl PCR reaction were added 4 units HaeIII, 1.5 units Sau3a or 5 units TaqI restriction enzyme. Restriction digestions were allowed to proceed for 60 min at 37° C. for HaeIII and Sau3A and at 65° C. for TaqI reactions. Restriction products were resolved in 1.5% agarose gels giving one restriction group for all inserts tested.

Five of the resulting, recombinant plasmids were selected for DNA sequencing. The inserts from three of the recombinant plasmids (called pT7PLR1-pT7PLR3) were generated by a combination of primers PLRN5 (SEQ ID NO:44) and PLRI5R (SEQ ID NO:46) with the 400 bp PCR product as substrate. The inserts from the remaining two recombinant plasmids (called pT7PLR4 and pT7PLR5) were generated from a combination of primers PLRN5 (SEQ ID NO:44) and PLRI4R (SEQ ID NO:45) and the 380 bp PCR product as substrate. All of the five, sequenced PCR products contained the same open reading frame.

The (+)-pinoresinol/(+)-lariciresinol reductase probe was constructed as follows: five, 100 μl PCR reactions were performed as described above with 10 ng pT7PLR3 DNA with primers PLRN5 (SEQ ID NO:44) and PLRI5R (SEQ ID NO:46). Gel-purified pT7PLR3 cDNA insert (50 ng) was used with Pharmacia's T7QuickPrime® kit and [α-³²P]dCTP, according to kit instructions, to produce a radiolabeled probe (in 0.1 ml), which was purified over BioSpin 6 columns (Bio-Rad) and added to carrier DNA (0.9 ml of 0.5 mg/ml sheared salmon sperm DNA obtained from Sigma).

Library Screening. 600,000 PFU of F. intermedia amplified cDNA library were plated for primary screening, according to Stratagene's instructions. Plaques were blotted onto Magna Nylon membrane circles (Micron Separations Inc.), which were then allowed to air dry. The membranes were placed between two layers of Whatman® 3MM Chr paper. cDNA library phage DNA was fixed to the membranes and denatured in one step by autoclaving for 2 min at 100° C. with fast exhaust. The membranes were washed for 30 min at 37° C. in 6×standard saline citrate (SSC) and 0.1% SDS and prehybridized for 5 h with gentle shaking at 57-58° C. in preheated 6×SSC, 0.5% SDS and 5×Denhardt's reagent (hybridization solution, 300 ml) in a crystallization dish (190×75 mm).

The [³²P]radiolabeled probe was denatured (boiling, 10 min), quickly cooled (ice, 15 min) and added to a preheated fresh hybridization solution (60 ml, 58° C.) in a crystallization dish (150×75 mm). The prehybridized membranes were next added to this dish, which was then covered with plastic wrap. Hybridization was performed for 18 h at 57-58° C. with gentle shaking. The membranes were washed in 4×SSC and 0.5% SDS for 5 min at room temperature, transferred to 2×SSC and 0.5% SDS (at room temperature) and incubated at 57-58° C. for 20 min with gentle shaking, wrapped with plastic wrap to prevent drying and finally exposed to Kodak X-OMAT AR film for 24 h at −80° C. with intensifying screens.

This screening procedure resulted in more than 350 positive plaques, with twenty (of different signal intensities) being subjected to two additional rounds of screening. After final purification, six of the twenty cDNAs were subcloned by in vivo excision into pBluescript. These six cDNAs were called plr-Fi1 to plr-Fi6 (SEQ ID Nos:47, 49, 51, 53, 55, 57).

In vivo Excision and Sequencing of plr-Fi1-plr-Fi6 Phagemids. The six purified cDNA clones were rescued from the phage following Stratagene's in vivo excision protocol. Both strands of the six different cDNAs (plr-Fi1 to plr-Fi6) that coded for (+)-pinoresinol/ (+)-lariciresinol reductase were completely sequenced using overlapping sequencing primers.

Purification of DNA for sequencing employed a QIAwell Plus plasmid purification system (QIAGEN) followed by PEG precipitation (Sambrook, J., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994)), with DNA sequences determined using an Applied Biosystems Model 373A automated sequencer. DNA and amino acid sequence analyses were performed using the Unix-based GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drive, Madison, Wis., USA 53711; Rice, P., Program Manual for the EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1Rq, England (1996)) and the ExPASy World Wide Web molecular biology server (Geneva University Hospital and University of Geneva, Geneva, Switzerland).

All six cDNAs had the same coding but different 5′-untranslated regions. On the other hand, analysis of the 3′-untranslated region of each of the six cDNAs established that all were truncated versions of the longest cDNA's 3′-region. Preliminary RNA gel blot analysis with total RNA from greenhouse-grown plant stem tips confirmed a single transcript with a length of approximately 1.2 kb.

RNA gel blot analysis. For RNA gel blot analysis, total RNA (30 μg per lane) from F. intermedia stem tips was separated by size by denaturing agarose gel electrophoresis. The RNA was transferred to charged nylon membranes (GeneScreen Plus®, Dupont NEN), cross-linked to the membrane (Stratalinker from Stratagene), prehybridized, hybridized with the same probe used to screen the cDNA library during cDNA cloning and washed according to the manufacturer's instructions for aqueous hybridization conditions. The membrane was then exposed to Kodak X-OMAT film for 48 hr at −80° C. with intensifying screens.

EXAMPLE 12 Expression of (+)-Pinoresinol/(+)-Lariciresinol Reductase cDNA plr-Fi1 (SEQ ID NO:47) in E. coli

Expression in Escherichia coli. In order to confirm that the putative (+)-pinoresinol/(+)-lariciresinol reductase cDNAs encoded functional (+)-pinoresinol/(+)-lariciresinol reductase, the cDNAs putatively encoding (+)-pinoresinol/(+)-lariciresinol reductase were heterologously expressed in E. coli. Heterologous expression was also necessary in order to obtain sufficient protein to enable the systematic study of the precise biochemical mechanism of (+)-pinoresinol/(+)-lariciresinol reductase at a future date.

Examination of the six putative (+)-pinoresinol/(+)-lariciresinol reductase clones revealed that one, plr-Fil (SEQ ID NO:47), was in frame with the α-complementation particle of β-galactosidase in pBluescript. This was fortuitous, since it potentially provided a facile means to express the fully functional fusion protein, and hence to provide proof that the cloned sequence was correct.

Purified plasmid DNA from plr-Fi1 (SEQ ID NO:47) was transformed into NovaBlue cells according to Novagen's instructions. Transformed cells (5 ml cultures) were grown at 37° C. with shaking (225 rpm) to mid log phase (OD₆₀₀=0.5) in LB medium (Sambrook, J., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994)) supplemented with 12.5 μg ml⁻¹ tetracycline and 50 μg ml⁻¹ ampicillin. IPTG (isopropyl β-D-thioglucopyranoside) was then added to a final concentration of 10 mM, and the cells were allowed to grow for 2 h. Cells were collected by centrifugation and resuspended in 500 μl (per 5 ml culture tube) buffer (20 mM Tris-HCl, pH 8.0, 5 mM dithiothreitol). Lysozyme (5 μl of 0.1 mg ml⁻¹, Research Organics, Inc.) was next added and following incubation for 10 min, the cells were lysed by sonication (3×15 s). After centrifugation at 14,000×g at 4° C. for 10 min, the supernatant was removed and assayed for (+)-pinoresinol/(+)-lariciresinol reductase activity (210 μl supernatant per assay) as described in Example 8.

Catalytic activity was established by incubating cell-free extracts for 2 h at 30° C. with (±)-pinoresinols (0.4 mM) and [4R-³H]NADPH (0.8 mM) under standard conditions. Following incubation, unlabeled (±)-lariciresinols and (±)-secoisolariciresinols were added as radiochemical carriers, with each lignan isolated by reversed-phase HPLC. Controls included assays of a pinoresinol/lariciresinol reductase cDNA which contains an out-of-frame cDNA insert, with all assay components, as well as plr-Fi1 (SEQ ID NO:47) and an out-of-frame pinoresinol/lariciresinol reductase cDNA with no substrate except [4R-³H]NADPH. Separation of products and chiral identification were performed by HPLC as previously described (Chu, A., et al., J. Biol. Chem. 268:27026-27033 (1993)).

Subsequent chiral HPLC analysis revealed that both (+)-lariciresinol and (−)-secoisolariciresinol, but not the corresponding antipodes, were radiolabeled (total activity: 54 nmol h⁻¹ mg⁻¹). By contrast, no catalytic activity was detected either in the absence of (±)-pinoresinols, or when control cells were used which contained a plasmid in which the cDNA insert was not in-frame with the β-galactosidase gene. Thus, the heterologously expressed (+)-pinoresinol/(+)-lariciresinol reductase and the plant protein function in precisely the same enantiospecific manner.

EXAMPLE 13 Sequence and Homology Analysis of the cDNA Insert of Clone plr-Fi1 (SEQ ID NO:47) Encoding (+)-pinoresinol/(+)-lariciresinol reductase

Sequence Analysis. The full length sequence of the cloned (+)-pinoresinol/(+)-lariciresinol reductase plr-Fi1 (SEQ ID NO:47) contained all of the peptide sequences determined by Edman degradation of digest fragments.

The single ORF predicts a polypeptide of 312 amino acids (SEQ ID NO:48) with a calculated molecular mass of 34.9 kDa, in close agreement with the value (˜35 or ˜36 kDa) estimated previously by SDS-PAGE for the two isoforms of (+)-pinoresinol/(+)-lariciresinol reductase. An equal number of acidic and basic residues are also present, with a theoretical isoelectric point (pI) of 7.09, in contrast to that experimentally obtained by chromatofocussing (pI˜5.7).

The amino acid composition reveals seven methionine residues. Interestingly, the N-terminus of the plant-purified enzyme lacks the initial methionine, this being the most common post-translational protein modification known. Consequently, the first methionine in the cDNA can be considered to be the site of translational initiation. The sequence analysis also reveals a possible N-glycosylation site at residue 215 (although no secretory targeting signal is present), and seven possible protein phosphorylation sites at residues 50 and 228 (protein kinase C-type), residues 228, 250, 302 and 303 (casein kinase II-type ) and residue 301 (tyrosine kinase type).

Regions of the pinoresinol/lariciresinol polypeptide chain (SEQ ID NO:48) were also identified that contained conserved sequences associated with NADPH binding (Jörnvall, H., in Dehydrogenases Requiring Nicotinamide Coenzymes (Jeffery, J., ed) pp. 126-148, Birkhäuser Verlag, Basel (1980); Branden, C., and Tooze, J., Introduction to Protein Structure, pp. 141-159, Garland Publishing, Inc., New York and London (1991); Wierenga, R. K. et al., J. Mol. Biol. 187:101-108 (1986)). There is a limited number of invariant amino acids in the sequences of different reductases which are viewed as indicative of NADPH binding sites. These include three conserved glycine residues with the sequence G-X-G-X-X-G (SEQ ID NO:76), where X is any residue, and six conserved hydrophobic residues. The glycine-rich region is considered to play a central role in positioning the NADPH in its correct conformation. In this regard, a comparison of the N-terminal region of (+)-pinoresinol/(+)-lariciresinol reductase with that of the conserved, NADPH-binding regions of Drosophila melanogaster alcohol dehydrogenase (Branden, C., and Tooze, J., Introduction to Protein Structure, pp. 141-159, Garland Publishing, Inc., New York and London (1991)), Pinus taeda cinnamyl alcohol dehydrogenase (MacKay J. J. et al., Mol. Gen. Genet. 247:537-545 (1995)), dogfish muscle lactate dehydrogenase (Branden, C., and Tooze, J., Introduction to Protein Structure, pp. 141-159, Garland Publishing, Inc., New York and London (1991)) and human erythrocyte glutathione reductase (Branden, C., and Tooze, J., Introduction to Protein Structure, pp. 141-159, Garland Publishing, Inc., New York and London (1991)), revealed some interesting parallels. The invariant glycine residues are aligned in every case, as are four of the six hydrophobic residues required for the correct packaging in the formation of the domain. Hence, the NADPH-binding site of (+)-pinoresinol/(+)-lariciresinol reductase isoforms is localized close to the N-terminus.

Homology Analysis: Comparison to Isoflavone Reductase. A BLAST search (Altschul, S. F, et al., J. Mol. Biol. 215:403-410 (1990)) was conducted with the translated amino acid sequence of (+)-pinoresinol/(+)-lariciresinol reductase (SEQ ID NO:48) against the non-redundant peptide database at the National Center for Biotechnology Information. Significant homology was noted for (+)-pinoresinol/(+)-lariciresinol reductase with various isoflavone reductases from the legumes, Cicer arietinum (Tiemann, K., et al., Eur. J. Biochem. 200:751-757 (1991)) (63.5% similarity, 44.4% identity), Medicago sativa (Paiva, N. L., et al., Plant Mol. Biol. 17:653-667 (1991)) (62.6% similarity, 42.0% identity) and Pisum sativum (Paiva, N. L., et al., Arch. Biochem. Biophys. 312:501-510 (1994)) (61.6% similarity, 41.3% identity). This observation is of considerable interest since isoflavonoids are formed via a related branch of phenylpropanoid-acetate pathway metabolism. Specifically, isoflavone reductases catalyze the reduction of α,β-unsaturated ketones during isoflavonoid formation. For example, the Medicago sativa L. isoflavone reductase catalyzes the stereospecific conversion of 2′-hydroxyformononetin to (3R)-vestitone in the biosynthesis of the phytoalexin, (−)-medicarpin (Paiva, N. L. et al., Plant Mol. Biol. 17:653-667 (1991)). This sequence similarity may be significant given that both lignans and isoflavonoids are offshoots of general phenylpropanoid metabolism, with comparable plant defense functions and pharmacological roles, e.g., as “phytoestrogens”. Consequently, since both reductases catalyze very similar reactions, it is tempting to speculate that the isoflavone reductases may have evolved from (+)-pinoresinol/(+)-lariciresinol reductase. This is considered likely since the lignans are present in the pteridophytes, hornworts, gymnosperms and angiosperms; hence their pathways apparently evolved prior to the isoflavonoids (Gang et al., In Phytochemicals for Pest Control, Hedin et al., eds, ACS Symposium Series, Washington D.C., 658:58-59 (1997)).

Comparable homology was also observed with putative isoflavone reductase “homologs” from Arabidopsis thaliana (Babiychuk, E., et al., Direct Submission (May 25, 1995) to the EMBL/GenBank/DDBJ databases (1995)) (65.9% similarity, 50.8% identity), Nicotiana tabacum (Hibi, N., et al., Plant Cell 6:723-735 (1994)) (64.6% similarity, 47.2% identity), Solanum tuberosum (van Eldik, G. J., et al., (1995) Direct submission (Oct. 6, 1995) to the EMBL/GenBank/DDBJ databases) (65.5% similarity, 47.7% identity) Zea mays (Petrucco, S., et al., Plant Cell 8:69-80 (1996)) (61.6% similarity, 44.9% identity) and especially Lupinus albus (Attuci, S., et al., Personal communication and direction submission (Jun. 6, 1996) to the EMBL/Genbank/DDBJ databases (1996)) (85.9% similarity, 66.2% identity).

By contrast, homology with other NADPH-dependent reductases was significantly lower: for example, dihydroflavonol reductases from Petunia hybrida (Beld, M. et al., Plant Mol. Biol. 13:491-502 (1989)) (43.2% similarity, 21.5% identity) and Hordeum vulgare (Kristiansen, K. N., and Rhode, W., Mol. Gen. Genet. 230:49-59 (1991)) (46.2% similarity, 21.1% identity), chalcone reductase from Medicago sativa (Ballance, G. M. and Dixon, R. A., Plant Physiol. 107:1027-1028 (1995)) (39.5% similarity, 15.8% identity), chalcone reductase “homolog” from Sesbania rostrata (Goormachtig, S., et al., (1995) Direct Submission (Mar. 13, 1995) to the EMBL/GenBank/DDBJ databases) (47.6% similarity, 24.1% identity), cholesterol dehydrogenase from Nocardia sp. (Horinouchi, S., et al., Appl. Environ. Microbiol. 57:1386-1393 (1991)) (46.6% similarity, 21.0% identity) and 3-β-hydroxy-5-ene steroid dehydrogenase from Rattus norvegicus (Zhao, H.-F., et al., Journal Endocrinology 127:3237-3239 (1990)) (43.5% similarity, 20.6% identity).

Thus, sequence analysis establishes significant homology between (+)-pinoresinol/(+)-lariciresinol reductase, isoflavone reductases and putative isoflavone reductase “homologs” which do not possess isoflavone reductase activity.

EXAMPLE 14 cDNA Cloning of Thuja plicata (−)-Pinoresinol/(−)-Lariciresinol Reductases

Plant Materials. Western red cedar plants (Thuja plicata) were maintained in Washington State University greenhouse facilities.

Materials. All solvents and chemicals used were reagent or HPLC grade. Taq thermostable DNA polymerase and restriction enzymes (SacI and XbaI) were obtained from Promega. pT7Blue T-vector and competent NovaBlue cells were purchased from Novagen and radiolabeled nucleotide ([α-³²P]dCTP) was purchased from DuPont NEN.

Oligonucleotide primers for polymerase chain reaction (PCR) and sequencing were synthesized by Gibco BRL Life Technologies. GENECLEAN II® kits (BIO 101 Inc.) were used for purification of PCR fragments, with the gel-purified DNA concentrations determined by comparison to a low DNA mass ladder (Gibco BRL) in 1.3% agarose gels.

Instrumentation. UV (including RNA and DNA determinations at OD₂₆₀) spectra were recorded on a Lambda 6 UV/VIS spectrophotometer. A Temptronic II thermocycler (Thermolyne) was used for all PCR amplifications. Purification of plasmid DNA for sequencing employed a QIAwell Plus plasmid purification system (Qiagen) followed by PEG precipitation (Sambrook, J., et al., Molecular Cloning. A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994)) or Wizard® Plus SV Minipreps DNA Purification System (Promega), with DNA sequences determined using an Applied Biosystems Model 373A automated sequencer.

Thuja plicata cDNA Library Synthesis. Total RNA (6.7 μg/g fresh weight) was obtained from young green leaves (including stems) of greenhouse-grown western red cedar plants (Thuja plicata) according to the method of Lewinsohn et al (Lewinsohn, E., et al., Plant Mol. Biol. Rep. 12:20-25 (1994)). A T. plicata cDNA library was constructed using 3 μg of purified poly(A)+mRNA (Oligotex-dT™ Suspension, Qiagen) with the ZAP-cDNA® synthesis kit, the Uni ZAP™ XR vector, and the Gigapack® II Gold packaging extract (Stratagene), with a titer of 1.2×10⁵ pfu for the primary library. The amplified library (7.1×10⁸ pfu /ml; 28 ml total) was used for screening (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994)).

T. plicata (−)-Pinoresinol/(−)-Lariciresinol Reductase cDNA Synthesis. T. plicata (−)-pinoresinol/(−)-lariciresinol reductase cDNA was obtained from mRNA by a reverse transcription-polymerase chain reaction (RT-PCR) strategy (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994)). First-strand cDNA was synthesized from the purified mRNA previously used for the synthesis of the T. plicata cDNA library, described above. Purified mRNA (150 ng) was mixed with linker-primer (1.4 μg) from ZAP-cDNA® synthesis kit (Stratagene), heated to 70° C. for 10 min, and quickly chilled on ice. The mixture of denatured mRNA template and linker-primer was then mixed with First Strand Buffer (Life Technologies), 10 mM DTT, 0.5 mM each dNTP, and 200 units of Super Script™II (Life Technologies) in a final volume of 20 μl. The reaction was carried out at 42° C. for 50 min and then stopped by heating (70° C., 15 min). E. coli RNase H (1.5 units, 1 μl) was added to the solution and incubated at 37° C. for 20 min.

The first-strand reaction (2 μl) was next used as the template in 100-μl PCR reactions (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂, 0.2 mM each dNTP, and 5 units of Taq DNA polymerase) with primer CR6-NT (5′GCACATAAGAGTATGGATAAG3′)(SEQ ID NO:60) (10 pmol) and primer XhoI-Poly(dT) (5′GTCTCGAGTTTTTTTTTTTTTTTTTT3′)(SEQ ID NO:59) (10 pmol). PCR amplification was carried out in a thermocycler as described in (Dinkova-Kostova, A. T., et al., J. Biol. Chem. 271:29473-29482 (1996)) except for the annealing temperature at 52° C. PCR products were resolved in 1.3% agarose gels, where at least two bands possessing the expected length (about 1,200-bp) were observed. The bands were extracted from the gel. The gel-purified PCR products (56 ng) were then ligated into the pT7Blue T-vector (50 ng) and transformed into competent NovaBlue cells, according to Novagen's instructions.

The size and orientation of the inserted cDNAs were determined using the rapid boiling lysis and PCR technique, following the manufacturer's (Novagen's) instructions, with the following primer combinations: R20-mer (SEQ ID NO:74) with U19-mer (SEQ ID NO:75); R20-mer (SEQ ID NO:74) with CR6-NT (SEQ ID NO:60); U19-mer (SEQ ID NO:75) with CR6-NT (SEQ ID NO:60). The CR6-NT primer end of the inserted DNAs was located next to the U19-mer primer site of the T-vector. The T-vectors containing the inserted cDNAs were purified with Wizard® Plus SV Minipreps DNA Purification System. Five inserted cDNAs were completely sequenced using overlapping sequencing primers and were shown to be identical except that polyadenylation sites were different. Therefore, the longest cDNA, designated plr-Tp1, (SEQ ID NO:61) was used for detection of enzyme activity using the pBluescript expression system.

Sequence Analysis—DNA and amino acid sequence analyses were performed using the Unix-based GCG Wisconsin Package (Program Manual for the Wisconsin Package, Version 8, September 1994, Genetics Computer Group, 575 Science Drives Madison, Wisconsin, USA 53711 (1996); Rice, P., Program Manual for the EGCG Package, Peter Rice, The Sanger Centre, Hinxton Hall, Cambridge, CB10 1Rq, England) and the ExPASy World Wide Web molecular biology server (Geneva University Hospital and University of Geneva, Geneva, Switzerland).

EXAMPLE 15 cDNA Cloning and Expression of Thuja plicata (+)-Pinoresinol/(+)-Lariciresinol Reductase

T. plicata (+)-Pinoresinol/(+)-Lariciresinol Reductase cDNA cloning. After plr-Tp1 (SEQ ID NO:61) was cloned and sequenced, the full-length clone was used to screen the T. plicata cDNA library as described in Example 11, except that the entire plr-Tp1 cDNA insert (SEQ ID NO:61) was used as a probe. Several positive clones were sequenced, revealing one new, unique cDNA which was called plr-Tp2 (SEQ ID NO:63). This cDNA encodes a reductase (SEQ ID NO:64) with high sequence similarity to plr-Tp1 (SEQ ID NO:62) (˜81% similarity at the amino acid level), but with substrate specificity properties identical to the original Forsythia intermedia reductase, as described below.

Enzyme Assays. Pinoresinol and lariciresinol reductase activities were assayed by monitoring the formation of [³H]lariciresinol and [³H]secoisolariciresinol as set forth in Example 8, with the following modifications. Briefly, each assay for pinoresinol reductase activity consisted of (±)-pinoresinols (5 mM in MeOH, 20 μl) and the enzyme preparation (i.e., total protein extract from E. coli, 210 μl). The enzymatic reaction was initiated by addition of [4R-³H]NADPH (10 mM, 6.79 kBq/mmol in distilled H₂O, 20 μl). After 3 hour incubation at 30° C. with shaking, the assay mixture was extracted with EtOAc (500 μl) containing (±)-lariciresinols (20 μg) and (±)-secoisolariciresinols (20 μg) as radiochemical carriers. After centrifugation (13,800×g, 5 min), the EtOAc solubles were removed and the extraction procedure was repeated. For each assay, the EtOAc solubles were combined with an aliquot (100 μl) removed for determination of its radioactivity using liquid scintillation counting. The remainder of the combined EtOAc solubles was evaporated to dryness in vacuo, reconstituted in MeOH/H₂O (30:70, 100 μl) and subjected to reversed phase and chiral column HPLC.

Lariciresinol reductase activity was assayed by monitoring the formation of (+)-[³H]secoisolariciresinol. These assays were carried out exactly as described above, except that (±)-lariciresinols (5 mM in MeOH, 20 μl) were used as substrates, with (±)-secoisolariciresinols (20 μg) added as radiochemical carriers.

Expression of plr-Tp1 (SEQ ID NO:61) in E. coli—In order for the open reading frame (ORF) of plr-Tp1 (SEQ ID NO:61) to be in frame with the β-galactosidase gene α-complementation particle in pBluescript SK(−), plr-Tp1 (SEQ ID NO:61) was excised out of pT7Blue T-vector with SacI and XbaI, gel-purified, and then ligated into the expression vector digested with these same enzymes. This plasmid, pPCR-Tp1, was transformed into NovaBlue cells according to Novagen's instructions. The transformed cells (5-ml cultures) were grown at 37° C. in LB medium (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 3 volumes, 3rd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1994)) supplemented with 50 μg ml⁻¹ carbenicillin with shaking (225 rpm) to mid log phase (A₆₀₀=0.5-0.7). The cells were next collected by centrifugation (1000 x g, 10 min) and resuspended in fresh LB medium supplemented with 10 mM IPTG (isopropyl β-D-thioglucopyranoside) and 50 μg ml⁻¹ carbenicillin to an absorbance of 0.6 (at 600 nm). The cells, allowed to grow overnight, were collected by centrifugation and resuspended in 500-700 μl of (per 5 ml culture tube) of buffer (50 mM Tris-HCl, pH 7.5, 2 mM EDTA, and 5 mM DTT). Next, the cells were lysed by sonication (5×45 s) and after centrifugation (17500×g, 4° C., 10 min) the supernatant was removed and assayed for (−)-pinoresinol/(−)-lariciresinol reductase activity as described above. Controls included assays of pBluescript (SK(−)) without insert DNA (as negative control) or with pPLR-Fi1 (SEQ ID NO:47) (cDNA of authentic F. intermedia (+)-pinoresinol/(+)-lariciresinol reductase in frame) as stereospecific control, as well as pPLR-Tp1 (SEQ ID NO:61) with no substrate except (4R)-³HNADPH.

The results showed that both (−)-lariciresinol and (+)-secoisolariciresinol were radiolabeled and that no incorporation of radioactivity was found in (−)-secoisolariciresinol. However, accumulation of radiolabel into (+)-lariciresinol was also observed, although at a much slower rate than that observed for (−)-lariciresinol. These results indicate that plr-Tp1(SEQ ID NO:62) can use both (−)-pinoresinol and (+)-pinoresinol as substrates, with the former being converted via (−)-lariciresinol completely to (+)-secoisolariciresinol, and the latter being converted much more slowly to (+)-lariciresinol, but not further to (−)-secoisolariciresinol.

Expression of plr-Tp2 (SEQ ID NO:63) in E. coli. The plr-Tp2 cDNA (SEQ ID NO:63) was found to be in frame with the β-galactosidase gene α-complementation particle in pBluescript SK(−). When evaluated for activity and substrate specificity, as described above, plr-Tp2 (SEQ ID NO:64) was found to possess the same substrate specificity and product formation as the original Forsythia intermedia reductase (Dinkova-Kostova, A. T., et al., J. Biol. Chem. 271:29473-29482 (1996)) except that a small amount of (−)-lariciresinol was also detected. This is interesting, because plr-Tp2 (SEQ ID NO:64) has a higher sequence similarity to plr-Tp1 (SEQ ID NO:62) than it does to the Forsythia reductase.

All the above observations were confirmed using deuterolabeled substrates (±)-[9,9′-²H₂, OC²H₃]pinoresinols with isolation of the corresponding lignans; each was then subjected to chiral column chromatography and HPLC-mass spectral analysis to confirm these findings.

EXAMPLE 16 Cloning of Additional Pinoresinol/Lariciresinol Reductases from Thuja plicata and Tsuga heterophylla

Two additional pinoresinol/lariciresinol reductases were cloned from a Thuja plicata young stem cDNA library as described in Example 15 for the cloning of plr-Tp2 (SEQ ID NO:63). The two additional pinoresinol/lariciresinol reductases were designated plr-Tp3 (SEQ ID NO:65) and plr-Tp4 (SEQ ID NO:67).

Two additional pinoresinol/lariciresinol reductases were cloned from a Tsuga heterophylla young stem cDNA library as described in Example 15 for the cloning of plr-Tp2 (SEQ ID NO:63). The two additional pinoresinol/lariciresinol reductases from Tsuga heterophylla were designated plr-Tp3 (SEQ ID NO:69) and plr-Tp4 (SEQ ID NO:71).

EXAMPLE 17

Cloning Additional Dirigent Protein cDNAs from Thuja plicata

An additional cDNA, called Tp9 (SEQ ID NO:77), encoding a dirigent protein (SEQ ID NO:78) was cloned from mRNA extracted from Western red cedar (Thuja plicata) in the following manner. First strand cDNA was synthesized from Western red cedar cambium RNA using primer CS1-895N (5′-AGAGTGGAGATTGTTGTCAAGAGTA-3′)(SEQ ID NO:79) derived from highly conserved sequence motifs found in other Western red cedar dirigent protein isoforms. SuperScript™II RNase H Reverse Transcriptase (Life Technologies, Rockville, Md.) was used to synthesize first strand cDNA following the manufacturer's protocol. Then the 3′ end of the first strand cDNA was tailed with dATP using terminal transferase (Boehringer Mannheim, Indianapolis, Ind.). The tailed cDNA was amplified via Expand™ High Fidelity PCR system (Boehringer Mannheim, Indianapolis, Ind.) using the following primers: first round of PCR utilized the oligo dT-anchor primer (5′-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTTTTV-3′)(SEQ ID NO:80)(wherein “V” at the 3′-end of the oligo-dT anchor primer represents A, C or G) as the 5′-sense primer, and primer CS1-895N (SEQ ID NO:79) as the 3′-antisense primer; second round of PCR utilized PCR anchor primer (5′-GACCACGCGTATCGATGTCGAC-3′)(SEQ ID NO:81) as the 5′-sense primer, and primer CS1-874N (5′-AGTAATAGGATCATCAAACAC-3′)(SEQ ID NO:82) as the 3′-antisense primer. The PCR conditions were as follows: first cycle, 94° C. for 1 minute; second thru twenty sixth cycles, 94° C. for 30 seconds, then 56° C. for 45 seconds, then 72° C. for two minutes; the twenty seventh cycle, 72° C. for seven minutes. The resulting PCR product (that corresponded to nucleic acid residues 1-253 of SEQ ID NO:77) was gel purified and cloned using a TA cloning kit (Invitrogen, Carlsbad, Calif.), and sequenced to identify the dirigent protein gene.

To complete the 3-region of the PCR product (that corresponded to nucleic acid residues 1-253 of SEQ ID NO:77), the oligo dT-anchor primer (SEQ ID NO:80) was used to generate first strand eDNA. The full length Tp9 cDNA (SEQ ID NO:77) was cloned using the following PCR primers: the first round of PCR utilized primer RT-CS-C1(−)50s (5′-CCAACTTCTTTCTCTACTTCAGAA-3′)(SEQ ID NO:83) as the 5′-sense primer, and the oligo dT anchor primer (SEQ ID NO:80) as the 3′-antisense primer; the second round of PCR utilized primer RT-CS-C1(−)31s (5′-CAGAACCCTGTTTTCTGATTTATT-3′)(SEQ ID NO:84) as the 5′-sense primer, and the PCR anchor primer (SEQ ID NO:81) as the 3′-antisense primer; and the third round of PCR utilized primer RT-CS-C1(−)13s (5′-TTTATTTTTGCACAATGGCAATCT-3′)(SEQ ID NO:85) as the 5′-sense primer, and the PCR anchor primer (SEQ ID NO:81) as the 3′-antisense primer. The reaction conditions for each round of PCR were as follows: first cycle, 94° C. for 1 minute; second thru twenty sixth cycles, 94° C. for 30 seconds, then 56° C. for 45 seconds, then 72° C. for two minutes; the twenty seventh cycle, 72° C. for seven minutes. The full length Tp9 cDNA sequence (SEQ ID NO:77) was confirmed by direct PCR amplification of the Tp9 gene using Expand™ High Fidelity PCR system.

EXAMPLE 18 Completing the 5′-Ends of Dirigent Protein Clones Tp3 (SEQ ID NO:24) and Tp4 (SEQ ID NO:26) from Thuja plicata

The nucleotide sequences of the Thuja plicata dirigent protein clones Tp3 (SEQ ID NO:24) and Tp4 (SEQ ID NO:26) were incomplete at their 5′ ends. Genomic DNA analysis indicated that Western red cedar dirigent protein genes do not contain introns. Consequently, it was possible to complete the partial cDNA sequences of Tp3 (SEQ ID NO:24) and Tp4 (SEQ ID NO:26) by utilizing the Advantage Genomic PCR Kit (Clonetech, Palo Alto, Calif.) following the manufacturer's instructions.

The cDNA clone (SEQ ID NO:86) encoding a full-length, Tp3 dirigent protein (SEQ ID NO:87) was cloned by PCR using Thuja plicata genomic DNA as template and the following primers: the first round of PCR utilized primer AP1 (5′-GTAATACGACTCACTATAGGGC-3′)(SEQ ID NO:88) as the 5′-sense primer, and primer TpS4-213n (5′-AGATTAGCTCCTTGAGGGGCTGAAACAA-3′)(SEQ ID NO:89) as the 3′-antisense primer; the second round of PCR utilized primer AP2 (5′-ACTATAGGGCACGCGTGGT-3′)(SEQ ID NO:90) as the 5′-sense primer, and primer TpS4-199n (5′-AGGGGCTGAAACAAGTGCAGATGTTGCA-3′)(SEQ ID NO:91) as the 3′-antisense primer; the third round of PCR utilized primer AP2 (SEQ ID NO:90) as the 5′-sense primer, and primer TpS4-188n (5′-CAAGTGTGCAGATGTTGCATTCTCTGCAT-3′)(SEQ ID NO:92) as the 3′-antisense primer.

The cDNA clone (SEQ ID NO:93) encoding a full-length, Tp4 dirigent protein (SEQ ID NO:94) was cloned by PCR using Thuja plicata genomic DNA as template and the following primers: the first round of PCR utilized primer AP1 (SEQ ID NO:88) as the 5′-sense primer, and primer CS10-826N (5′-CAGTCATAATGGTGAGATTGGCTCCCT-3′)(SEQ ID NO:95) as the 3′-antisense primer; the second round of PCR utilized primer AP2 (SEQ ID NO:90) as the 5′-sense primer, and primer CS10-814N (5′-TGAGATTGGCTCCCTCAGGGGCTGCAA-3′)(SEQ ID NO:96) as the 3′-antisense primer; the third round of PCR utilized primer AP2 (SEQ ID NO:90) as the 5′-sense primer, and primer CS10-795N (5′-GGCTGCAACAAGTGCAGATGTTGCATT-3′)(SEQ ID NO:97) as the 3′-antisense primer.

The PCR reaction conditions for cloning the full-length, Tp3 and Tp4 dirigent protein clones having the nucleic acid sequences set forth in SEQ ID NO:86 and SEQ ID NO:93 were identical and were as follows: the first round of PCR included primary PCR consisting of a first cycle at 94° C. for 1 minute and second thru eighth cycles at 94° C. for 25 seconds, then 72° C. for 12 minutes, followed by secondary PCR consisting of a first cycle at 94° C. for 1 minute, second thru thirty three cycles at 94° C. for 20 seconds, then 72° C. for 6 minutes, and finally a thirty fourth cycle at 68° C. for 12 minutes; the second and third rounds of PCR each consisted of a first cycle at 94° C. for one minute, then second thru twenty sixth cycles at 94° C. for 30 seconds, then 60° C. for 45 seconds, then 72° C. for two minutes, then a twenty seventh cycle at 72° C. for seven minutes.

The ability to complete the full-length Tp3 (SEQ ID NO:86) and Tp4 (SEQ ID NO:93) sequences from genomic DNA overcomes the difficulty in completing a full-length sequence from a cDNA pool. Incomplete synthesis of the first strand cDNA of target cDNAs often occurs due to instability of RNA or secondary structure of RNA, and results in the incomplete synthesis of the first strand cDNA.

EXAMPLE 19 Cloning a Dirigent Protein cDNA from Eucommia ulmoides

A cDNA (SEQ ID NO:98) encoding a dirigent protein (SEQ ID NO:99) was isolated from Eucommia ulmoides utilizing the strategy used for the cloning of the Tp9 cDNA (SEQ ID NO:77) of Western red cedar. First strand cDNA was synthesized with SuperScript™II RNase H Reverse Transcriptase from E. ulmoides leaf RNA using the oligo dT anchor primer (SEQ ID NO:80). From the first strand cDNA, a 300 bp cDNA fragment (corresponding to nucleotides 152 thru 452 of SEQ ID NO:98) was amplified using N-terminal primer 5′-GARTTGGTGTTCTATTTCCACGACATMC-3′ (SEQ ID NO:100)(wherein “R” represents A or G and “M” represents A or C) and C-terminal primer 5′-CAAAGTGGCAACCCCTGTCGCCATG-3′ (SEQ ID NO:101), derived from a highly conserved sequence among the other dirigent protein isoforms. The PCR reaction conditions were as follows: first cycle, 94° C. for lmin, second thru twenty sixth cycles, 94° C. for 30 seconds, then 50° C. for 45 seconds, then 72° C. for 2 min, and a twenty seventh cycle at 72° C. for 7 min.

The PCR product was gel purified and cloned using a TA cloning kit. Then this fragment was used to design gene specific primers to clone a complete sequence. In order to clone the missing 5′ region, first strand cDNA was synthesized with SuperScript™II RNase H^(—)Reverse Transcriptase using the gene specific, 3′-antisense Sp2 primer (5′-CCCCCGTTCCTCCAACCACCGG-3′)(SEQ ID NO:102), and the 3′ end of the first strand cDNA was tailed with dATP using terminal transferase. The tailed cDNA was amplified via Expand TM High Fidelity PCR system using the oligo dT-anchor primer (SEQ ID NO:80) and the Sp2 primer (SEQ ID NO:102). Then the second round of PCR was conducted using the PCR-anchor primer (SEQ ID NO:81) and the Sp2 primer (SEQ ID NO:102). The PCR product was gel purified, and cloned using the TA cloning kit. The resulting, partial cDNA corresponded to nucleic acid residues 1 thru 413 of SEQ ID NO:98. The PCR reaction conditions were the same for the first and second rounds of PCR and were as follows: first cycle, 94° C. for 1 min, second thru twenty sixth cycles, 94° C. for 30 seconds, then 50° C. for 45 seconds, then 72° C. for 2 min, and a twenty seventh cycle at72° C. for 7 min.

To obtain the missing 3′ sequence, the oligo dT-anchor primer (SEQ ID NO:80) was used to generate first strand cDNA. The full length Eucommia cDNA (SEQ ID NO:98) was amplified through two consecutive rounds of PCR using a set of nested primers: 5′-sense primers SpN1 (5′-GGCCCATGCGGTTAAGCATATTCTCC-3′)(SEQ ID NO:103) and SpN2 (5′-CCTCTATAAAAACATAATTCTTTTCCCCC-3′)(SEQ ID NO:104), and the primers having the nucleic acid sequences set forth in SEQ ID NO:80 and SEQ ID NO:81 as 3′-antisense primers. The reaction conditions for both the first and the second rounds of PCR were as follows: first cycle, 94° C. for 1 minute, second thru twenty sixth cycles, 94° C. for 30 seconds, then 60° C. for 45 seconds, then 72° C. for 2 minutes, a twenty seventh cycle at 72° C. for 7 minutes.

The PCR product was gel purified, and cloned using a TA cloning kit. The identity of the full length cDNA (SEQ ID NO:98) was confirmed by sequencing and by direct PCR amplification of the corresponding Eucommia dirigent protein gene.

EXAMPLE 20 Cloning a Dirigent Protein cDNA from Schisandra chinensis

A cDNA (SEQ ID NO:105), encoding a dirigent protein (SEQ ID NO:106) was cloned from mRNA extracted from Schisandra chinensis in the following manner. A cDNA library was made from Schisandra chinensis leaf and stem tissues. The cDNA library was probed for a homologous dirigent protein clone using the ³²P-CTP-labelled F. intermedia dirigent protein gene (Psd-Fi1) (SEQ ID NO:12) as a probe. Three rounds of screening were utilized. For each screen, plaques were lifted onto nylon transfer membrane for 3 minutes, the transferred DNA was then fixed to the membrane by autoclaving for 3 minutes at 100° C., and debris were washed off the membranes with 2×SSC for 30 minutes at 37° C. Prehybridization solution consisted of 6×SSC, 5×Denhardt's reagent, and 0.5% SDS. 50 μl probe (SEQ ID NO:12) was made with T7 Quick-Prime Kit. 100 μl 5 mg/ml salmon sperm DNA and 850 μl deionized, distilled water were then added to the probe. Hybridization solution consisted of 6×SSC, 5×Denhardt's reagent, 0.5% SDS, and probe (SEQ ID NO:12).

For the primary screen, nylon membrane filters bearing plaques were prehybridized for seven and a half hours at 48-49° C. The filters were then hybridized overnight (approximately 17 hours) at 49° C. in the presence of 1.13×10⁷ cpm radiolabelled probe (SEQ ID NO:12). After hybridization, the filters were washed with 4×SSC, 0.5% SDS, for 10 min, then with 2×SSC, 0.5% SDS, for 9 min at room temperature (from about 20° C. to about 24° C.), then with 2×SSC, 0.5% SDS, for 50 minutes, warming from room temperature (from about 20° C. to about 24° C.) to 49° C. Finally the filters were rinsed with 6×SSC briefly before being exposed to film for one and a half days.

For the secondary screen, nylon membrane filters bearing plaques were prehybridized for 8 hours at 49° C. The filters were then hybridized overnight (approximately 16 hours) at 49° C. in the presence of 3.9×10⁶ cpm radiolabelled probe (SEQ ID NO:12). After hybridization, the filters were washed with 4×SSC, 0.5% SDS, at room temperature for 5-20 minutes, then rinsed briefly in 6×SSC before being exposed to film overnight.

For the tertiary screen, nylon membrane filters bearing plaques were prehybridized overnight at 45° C. The filters were then hybridized overnight (approximately 18 hours) at 45° C. in the presence of 1.2×10⁶ cpm radiolabelled probe (SEQ ID NO:12). After hybridization, the filters were washed with 4×SSC, 0.5% SDS, for 10 minutes, then with 2×SSC, 0.5% SDS, for 2 minutes at room temperature (from about 20° C. to about 24° C.). Finally the filters were rinsed with 6×SSC briefly before being exposed to film for 4 days.

After three rounds of screening, one positive plaque was isolated. The cDNA from this plaque was subsequently excised and cloned into SOLR cells (Stratagene Cloning Systems, 11011 North Torrey Pines Road, La Jolla, Calif. 92037). Sequence analysis showed it to be a full-length clone, and homology analysis established it to be 51-72% identical and 63-80% similar to other known dirigent proteins at the amino acid level.

EXAMPLE 21 Cloning a Pinoresinol/Lariciresinol Reductase cDNA from Linum usitatissimum

A cDNA (SEQ ID NO:107), encoding a pinoresinol/lariciresinol reductase protein (SEQ ID NO:108), was cloned from mRNA extracted from Linum usitatissimum (Flax) in the following manner. Total RNA was obtained from two week old whorls (each containing 10 developing flax seeds) of green house grown L. usitatissimum plants. Poly(A)⁺mRNA was purified using the Promega PolyATract® mRNA Isolation System. A L. usitatissimum seed cDNA library was constructed using 5 μg of purified mRNA with the Stratagene ZAP-cDNA® Synthesis Kit and the ZAP-cDNA® Gigapack® III Gold cloning kit, with a titer of 2.8×10⁵ pfu (plaque forming units) for the primary library. Approximately 35 ml of liquid phage lysate from the amplified library (2.8×10⁹ pfu/ml) was used to obtain pure cDNA library DNA for PCR screening.

The following degenerate primers were designed based on N-terminal, C-terminal and internal regions of similarity found in the Forsythia intermedia and Thuja plicata (Western red cedar) pinoresinol/lariciresinol reductase clones: PLR4 forward primer (5′-CCITCIGAGTTCGGIATGGATCCI-3′)(SEQ ID NO:109), and PLR6 reverse primer (5′-IGTATATTTIACTTCIGGGTA-3′)(SEQ ID NO:110). Pure L. usitatissimum cDNA library DNA (˜5 ng) was used as the template in 45 μl PCR reactions with various primer combinations. PCR amplification was carried out in an Amplitron® II thermocycler as follows: 5 cycles of 1 minute at 94° C., then 1 minute at 37° C., then 3 minutes at 72° C., followed by 25 cycles of 25 seconds at 94° C., then I minute at 48° C., and 2 minutes at 72° C.; with 10 minutes at 72° C. and an indefinite hold at 4° C. after the final cycle. PCR products were resolved in 1% agarose gels. Reactions producing single bands with an accurate size predicted by their respective primer combination were cloned using Invitrogen's Topo-TA cloning® Kit and sequenced. This round of PCR yielded a partial-length cDNA clone of about 600 bp (SEQ ID NO:111).

The partial length clone (SEQ ID NO:111) was used as a ³²P-radiolabeled probe with the aim of isolating the full length sequence. 500,000 pfu of L. usitatissimum amplified cDNA library were plated, plaques were blotted onto Magna Nylon membrane circles, and the library was screened as follows. The blotted membranes were placed between two layers of Whatman 3MM Chr. Paper. cDNA library phage DNA was fixed to the membranes and denatured in one step by autoclaving for 4 min at 100° C. with fast exhaust. The membranes were washed for 30 min with gentle shaking at 37° C. in 6×SSC and 0.1% SDS, and prehybridized for 5 h with gentle shaking at 57° C. in preheated 6×SSC, 0.5% SDS, and 5×Denhardt's reagent (hybridization solution, 220 ml) in a crystallization dish (190×75 mm).

The ³²P-radiolabeled probe (SEQ ID NO:111) was denatured (98° C. for 10 minutes), quickly cooled (on ice for 15 minutes), and added to a preheated hybridization solution (50 ml at 57° C.) in a crystallization dish (150×75 mm). The prehybridized membranes were next added to this dish, which was then covered with plastic wrap. Hybridization was performed for 17 h at 57° C. with gentle shaking. The membranes were washed in 4×SSC and 0.5% SDS (250 ml) for 5 min at room temperature, transferred to preheated 2×SSC and 0.5% SDS (250 ml), and incubated at 57° C. for 20 min with gentle shaking. After the membranes were removed from the dish and wrapped with plastic wrap, they were exposed to Kodak X-OMAT AR film for 24 h at −80° C. between intensifying screens. A larger cDNA clone (SEQ ID NO:112) was attained, but in comparison to F. intermedia cDNA plr-Fi1 (SEQ ID NO:47) it still lacked ˜258 bp at the N-terminal.

To achieve a full length clone, gene specific primer 1 (5′-AACATTTCCGGCCTCTTTGATGGCCTCGAC-3′)(SEQ ID NO:113) and gene specific primer 2 (5′-AAGGTAGATCATCAGATAATCTTTCATACG-3′)(SEQ ID NO:114) were designed and used in combination with the Stratagene Uni-ZAP® XR vector T7 (5′-GTAATACGACTCACTATAGGGC-3′)(SEQ ID NO:115) and T3 (5′-AATTAACCCTCACTAAAGGG-3′)(SEQ ID NO:116) primers. A 936 bp gene (SEQ ID NO:107), containing identical sequence information from all previous truncated clones, displayed an ˜74% similarity and ˜61% identity to F. intermedia cDNA plr-Fi1 (SEQ ID NO:47) at the amino acid level.

EXAMPLE 22 Cloning a Pinoresinol/Lariciresinol Reductase cDNA from Schisandra chinensis

A cDNA (SEQ ID NO:117), encoding a pinoresinol/lariciresinol reductase protein (SEQ ID NO:118) was cloned from mRNA extracted from Schisandra chinensis in the following manner. Degenerate primers PLR4 (SEQ ID NO:109) and PLR6 (SEQ ID NO:110) were made from regions of high homology between known reductase clones, and these were used to screen a Schisandra chinensis leaf and stem tissue cDNA library using a PCR-guided strategy. The Schisandra chinensis leaf cDNA library was first used as a template under the following conditions: five cycles of 95° C. for 1 minute, 37° C. for 1 minute and 72° C. for 3 minutes, then 25 cycles of 94° C. for 25 minutes, 48° C. for 1 minute and 72° C. 2 minutes. No cDNA bands were found upon gel analysis of the resulting products which were then used as template for another round of PCR under the following conditions: five cycles of 95° C. for 1 minute, 37° C. 1 minute, 72° C. 3 minutes, then 25 cycles of 94° C. for 25 minutes, 48° C. for 1 minute, 72° C. for 2 minutes.

A 600 bp PCR product was obtained, which was then cloned into a TOPO-TA vector and partially sequenced to yield the sequence of SEQ ID NO:119. Sequence analysis showed it to be 55.7% identical and 67.0% similar to F. intermedia cDNA plr-Fi1 (SEQ ID NO:47) at the amino acid level. This PCR product (including the nucleic acid sequence set forth in SEQ ID NO:119) was then used to make a ³²P-CTP probe, and the cDNA library was once again probed using three rounds of screening.

For the primary screen, cDNA library plaques were transferred onto nylon transfer membrane for 3 minutes, then fixed to the membranes by autoclaving for 3 minutes at 100° C. Debris was washed off membranes with 2×SSC for 30 minutes at 37° C., then the membranes were incubated in prehybridization solution consisting of 6×SSC, 5×Denhardt's reagent, and 0.5% SDS, for 6 hours at 48° C. The filters were then hybridized for 24 hours at 45° C. in hybridization solution (6×SSC, 5×Denhardt's reagent, 0.5% SDS), including 100 μl 5 mg/ml salmon sperm DNA, 850 μl ddH₂O and 9.6×10⁵ cpm probe. The filters were then washed (at room temperature) with 4×SSC, 0.5% SDS, for 5 minutes, then with 2×SSC, 0.5% SDS, for 20 minutes, then rinsed briefly with 4×SSC and exposed to autoradiography film 5 days.

The secondary screen was conducted under the same conditions as the primary screen except that the filters were prehybridized for 24 hours at 48° C., then hybridized for 24 hours at 48° C. in the presence of 1.37×10⁶ cpm probe. The filters were washed at room temperature with 4×SSC, 0.5% SDS, for 5 minutes then briefly rinsed in 6×SSC at room temperature and exposed to film overnight.

The tertiary screen was conducted under the same conditions as the primary screen except that the filters were prehybridized for 24 hours at 48° C., then hybridized for 37 hours at 48° C. in the presence of 2.2×10⁶ cpm probe. The filters were washed at room temperature under the following conditions: 4×SSC, 0.5% SDS, for 5 minutes, then with 2×SSC, 0.5% SDS, for 5 minutes, rinsed briefly in 6×SSC and exposed to film overnight.

One positive plaque was identified and isolated after the foregoing three rounds of screening. The plaque was excised into a pBluescript plasmid, cloned into SOLR cells, and sequenced (SEQ ID NO:119). Sequence analysis showed this clone (SEQ ID NO:119) encoded a partial length reductase protein (SEQ ID NO:120) which was 57.4% identical and 78.7% similar to F. intermedia PLR at the peptide level. However, a one-base frame-shift mutation (deletion of an adenine at position 579) was present in the full-length clone (SEQ ID NO:119) which prevented expression of full-length protein.

This full-length clone (SEQ ID NO:119) was next used to make a ³²P-dCTP probe, and the library was further screened in the following manner. For the primary screen, cDNA library plaques were transferred onto nylon transfer membrane for 3 minutes, then fixed to the membranes by autoclaving for 3 minutes at 100° C. Debris was washed off membranes with 2×SSC for 30 minutes at 37° C., then the membranes were incubated in prehybridization solution consisting of 6×SSC, 5×Denhardt's reagent, and 0.5% SDS, for 18 hours at 47° C. The filters were then hybridized for 24 hours at 47° C. in hybridization solution (6×SSC, 5×Denhardt's reagent, 0.5% SDS), including 100 μl 5 mg/ml salmon sperm DNA, 850 μl deionized, distilled H₂O and 9.3×10⁶ cpm probe. The filters were then washed at room temperature with 4×SSC, 0.5% SDS, for 5-10 minutes, then rinsed briefly at room temperature with 6×SSC and exposed to autoradiography film overnight.

The secondary screen was conducted under the same conditions as the primary screen except that the filters were prehybridized for eight and a third hours at 45-47° C., then hybridized overnight at 40-47° C. in the presence of 7.5×10⁶ cpm probe. The filters were washed with 4×SSC, 0.5% SDS, for 10 minutes, then with 2.5×SSC, 0.5% SDS for 10 minutes at room temperature, then washed with 2.5×SSC, 0.5% SDS, for 10 minutes with the temperature rising to 42° C. during that period, and finally rinsing in 6×SSC briefly at room temperature and exposed to film overnight.

The tertiary screen was conducted under the same conditions as the primary screen except that the filters were prehybridized for 11 hours at 47° C., then hybridized overnight (approximately 14 hours) at 47° C. in the presence of 1.8×10⁶ cpm probe. The filters were washed at room temperature with 4×SSC, 0.5% SDS, for 10 minutes, then rinsed at room temperature in 6×SSC briefly and exposed to film for approximately 24 hours.

Two positive plaques were identified after the foregoing three rounds of screening. Each was excised, transformed into SOLR cells, and sequenced. Of these, one clone (SEQ ID NO:117) was a full-length reductase identical to the clone having the sequence set forth in SEQ ID NO:119, with the exception that it was in frame. The other clone was not full-length. The full-length reductase cDNA (SEQ ID NO:119) was cloned into a pBad-TOPO-TA expression vector.

Based on the characterization and sequence comparison of the foregoing pinoresinol/lariciresinol reductase cDNAs and proteins, presently preferred pinoresinol/lariciresinol reductase proteins of the invention utilize NADPH as a cofactor and include the conserved amino acid sequence domain Gly Xaa Gly Xaa Xaa Gly (SEQ ID NO:76).

EXAMPLE 23 Cloning Additional Nucleic Acid Molecules that Encode a Dirigent Protein

Additional nucleic acid molecules encoding a dirigent protein can be cloned utilizing a variety of strategies. In one approach, genomic DNA or cDNA can be amplified by PCR using the following primer pair: primer PS-6For 5′-KGTGTTYGAYGATCCYATTACYBTWGACAAC-3′ (SEQ ID NO:120) and primer PS-2Rev 5′-TGRCTAMGTAWACTYCCTCTACAAATAAAG-3′ (SEQ ID NO:121). The sequence of PCR reactions is set forth in Table 4 below. Representative PCR reaction conditions are as set forth in Table 6 below, except that primers PS-6For (SEQ ID NO:120) and PS-6Rev (SEQ ID NO:121) are utilized instead of primers PLR4 forward (SEQ ID NO:109) and PLR6 reverse (SEQ ID NO:110).

TABLE 4 I Temp. (° C.) Time 94 1 min 60 2 min 72 3 min 35 cycles 72 10 min 

The resulting nucleic acid molecule(s) can be used to screen a cDNA library or genomic DNA library to isolate a full-length, or substantially full-length, dirigent protein cDNA or gene. Representative library screening conditions are hybridization in 6×SSC, 5×Denhardt's, 0.5% SDS at 55-58° C. for 12 hours, followed by washing in 2×SSC, 0.5% SDS at 55-58° C. for 30 minutes. An optional further wash can be conducted in 1×SSC, 0.5% SDS at 55-58° C. for 30 minutes, followed, if so desired, by an additional, optional, wash in 0.5×SSC, 0.5% SDS at 55-58° C. for 30 minutes.

In another approach, genomic DNA molecules encoding a dirigent protein can be cloned by PCR utilizing either primer PS-6For (SEQ ID NO:120) or primer PS-2Rev (SEQ ID NO:121) and an adapter primer that is complementary to one strand of an oligonucleotide adapter ligated to the ends of the genomic DNA fragments that form the target population of nucleic acid molecules in the PCR reaction. Adapter molecules are well known to those of ordinary skill in the art and are described, for example, in Molecular Cloning, A Laboratory Manual (2^(nd) edition), Sambrook et al (eds), Chapter 8, which chapter is incorporated herein by reference. Table 5 sets forth three, representative PCR cycling regimes that can be utilized in this aspect of the invention to clone genomic DNA molecules encoding a dirigent protein. Representative PCR reaction conditions are set forth in Table 6, except that primers PS-6For (SEQ ID NO:120) or primer PS-2Rev (SEQ ID NO:121) and an adapter primer are utilized instead of primers PLR4 forward (SEQ ID NO:109) and PLR6 reverse (SEQ ID NO:110).

TABLE 5 Temp. (° C.) Time I 94  1 min 60  2 min 72  3 min 35 cycles 72 10 min II 94 20 sec 72  3 min 7 cycles 94 20 sec 67  3 min 32 cycles 67  7 min III 94 15 sec 72 12 min 7 cycles 94 20 sec 68  6 min 32 cycles 67  6 min

In yet another approach, nucleic acid molecules encoding a dirigent protein can be cloned by PCR utilizing genomic DNA or cDNA as template and pools of degenerate primers. Exemplary PCR cycling regimes useful in this aspect of the invention are those set forth as Program I in Table 5 above, except that the annealing temperature is 45° C. to 50° C. Representative PCR reaction conditions are set forth in Table 6, except that pools of degenerate primers are utilized instead of primers PLR4 forward (SEQ ID NO:109) and PLR6 reverse (SEQ ID NO:110).

In a further approach, cDNA molecules encoding a dirigent protein can be cloned by initially cloning a target population of cDNA molecules into a vector that includes binding sites for the T3 primer (SEQ ID NO:116) and the T7 primer (SEQ ID NO:117) flanking each cDNA insert. The cloned population of cDNA molecules is then utilized as template in a PCR reaction that utilizes either primer PS-6For (SEQ ID NO:120) or primer PS-2Rev (SEQ ID NO:121) and either the T3 primer (SEQ ID NO:116) or the T7 primer (SEQ ID NO:117). A representative PCR cycling regime is set forth as Program I in Table 5 above. Representative PCR reaction conditions are set forth in Table 6, except that primers PS-6For (SEQ ID NO:120) or primer PS-2Rev (SEQ ID NO:121) and either the T3 primer (SEQ ID NO:116) or the T7 primer (SEQ ID NO:117) are utilized instead of primers PLR4 forward (SEQ ID NO:109) and PLR6 reverse (SEQ ID NO:110).

Thus, in one aspect, the present invention provides isolated nucleotide sequences encoding dirigent proteins, the nucleotide sequences being capable of remaining hybridized to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 77, 86, 93, 98 and 105 under wash conditions of 1×SSC at 58° C. The present invention also provides vectors, such as replicable expression vectors, that include one or more nucleic acid molecules of the invention, and host cells that include one or more vectors of the invention.

The present invention also provides methods of enhancing the expression of dirigent protein in a host cell. The methods of enhancing the expression of dirigent protein in a suitable host cell include the steps of (a) introducing into the host cell a replicable expression vector that comprises a nucleic acid sequence encoding a dirigent protein, the nucleic acid sequence being capable of remaining hybridized to the antisense complement of a nucleic acid sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 77, 86, 93, 98 and 105 under stringent wash conditions, and (b) expressing the encoded dirigent protein.

The present invention also provides methods of inhibiting the expression of dirigent protein in a host cell. The methods include the steps of (a) introducing into the host cell a replicable vector that includes a nucleic acid sequence capable of hybridizing to a nucleic acid sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 77, 86, 93, 98 and 105 under stringent wash conditions, and (b) transcribing the nucleic acid sequence.

In another aspect, the present invention provides methods of producing optically-pure lignans. The methods include the steps of (a) introducing into a host cell an expression vector that includes a nucleic acid sequence encoding a dirigent protein capable of directing a bimolecular phenoxy coupling reaction to produce an optically pure lignan, the nucleic acid sequence being capable of remaining hybridized to the antisense complement of a nucleic acid sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 77, 86, 93, 98 and 105 under stringent wash conditions, (b) expressing the encoded dirigent protein and (c) purifying optically pure lignan from the host cell.

EXAMPLE 24 Cloning Additional Nucleic Acid Molecules That Encode a Pinoresinol/Lariciresinol Reductase

Additional nucleic acid molecules encoding a pinoresinol/lariciresinol reductase can be cloned utilizing a variety of strategies. In one approach, a DNA probe is generated from cDNA or genomic DNA by PCR, and the probe is then used to screen a cDNA or genomic DNA library. Representative PCR reaction conditions are set forth in Table 6 below. The primers utilized are PLR4 forward (SEQ ID NO:109) and PLR6 reverse (SEQ ID NO:110)

TABLE 6 Ingredients Volume (μl) Supplier H₂O 27.75 25 mM MgCl₂ 5.0 Fischer Scientific 10 X assay buffer: 5.0 Fischer Scientific 100 mM Tris-HCl pH 8.3 (25° C.) 500 mM KCl Taq DNA polymerase 0.125 Fischer Scientific dNTPs (20 mM) 0.5 Sigma 10 X PLR4 forward 5.0 Gibco BRL Life Technologies (10 pmol/μl) (SEQ ID NO: 109) 10 X PLR6 reverse 5.0 Gibco BRL Life Technologies (10 pmol/μl) (SEQ ID NO: 110) Genomic/cDNA template X (5 ng) Total volume 45.0

A representative PCR thermocycler program useful in this aspect of the invention is set forth in Table 7.

TABLE 7 Temp. (° C.) Time 94 1 min 37 1 min 72 3 min 5 cycles 94 25 sec  48 1 min 72 2 min 25 cycles 72 10 min   4 hold

The DNA fragment generated by the foregoing PCR reactions can be used as a probe to screen a cDNA or genomic DNA library in order to isolate a full-length, or substantially full-length, pinoresinol/lariciresinol reductase clone. Representative hybridization conditions are hybridization in 6×SSC, 5×Denhardt's and 0.5% SDS at 57° C. Representative wash conditions are one wash in 4×SSC, 0.5% SDS, at room temperature (typically 20° C. to 30° C.) for 5 minutes, followed by one wash in 2×SSC, 0.5% SDS at 57 ° C. for 20 minutes. An optional further wash can be conducted in 1×SSC, 0.5% SDS at 57° C. for 30 minutes, followed, if so desired, by an additional, optional, wash in 0.5×SSC, 0.5% SDS at 57° C. for 30 minutes.

In another approach, DNA molecules encoding a pinoresinol/lariciresinol reductase protein can be cloned from mRNA by RT-PCR, i.e., a PCR reaction that utilizes mRNA as the initial substrate. First-strand DNA is synthesized from the purified mRNA of interest. Purified mRNA (150 ng) is mixed with 1.4 μg linker-primer (5′-CTCGAGTTTTTTTTTTTT-3′) (SEQ ID NO:122) from ZAP-cDNA® synthesis kit (Stratagene), heated to 70° C. for 10 min, and quickly chilled on ice. The mixture of denatured mRNA template and linker-primer (SEQ ID NO:122) is then mixed with First Strand Buffer (Life Technologies), 10 mM DTT, 0.5 mM each dNTP, and 200 units of SuperScript™ II (Life Technologies) in a final volume of 20 μl. The reaction is carried out at 42° C. for 50 minutes and then stopped by heating (70° C., 15 min). E. coli Rnase H (1.5 units, 1 μl) is added to the solution and incubated at 37° C. for 20 min.

The first-strand reaction (2 μl) is next used as the template in 100-μl PCR reactions (10 mM Tris-HCl, pH 9.0, 50 mM KCl, 0.1% Triton X-100, 1.5 mM MgCl₂, 0.2 mM each dNTP, and 5 units of Taq DNA polymerase) with primer CR6-NT (5′GCACATAAGAGTATGGATAAG3′) (SEQ ID NO:60) (10 pmol) and primer XhoI-Poly(dT) (5′GTCTCGAGTTTTTTTTTTTTTTTTTT3′)(SEQ ID NO:59)(10 pmol). PCR amplification is carried out in a thermocycler as described in Dinkova-Kostova, A. T., et al., J. Biol. Chem. 271:29473-29482 (1996)(which publication is incorporated herein by reference), except for the annealing temperature at 52° C. In brief, the PCR amplification regime includes: 35 cycles, each cycle including one minute at 94° C., two minutes at 52° C., and three minutes at 72° C.; followed by 5 minutes at 72° C. and an indefinite hold at 4° C. after the final cycle. PCR products are resolved in 1.3% agarose gels to reveal a band of about 1,200-bp. The gel-purified PCR product (˜50 ng) is then ligated into the pT7Blue T-vector (50 ng) and transformed into competent NovaBlue cells, according to Novagen's instructions. The inserted cDNA is next sequenced using overlapping sequencing primers.

The cDNA so obtained can be used to screen cDNA or genomic DNA libraries for full-length, or substantially full-length, pinoresinol/lariciresinol reductase clones. Representative hybridization conditions for screening cDNA or genomic DNA libraries are 6×SSC, 5×Denhardt's, 0.5% SDS at 57-58° C. Representative wash conditions are one wash in 4×SSC, 0.5% SDS at room temperature (typically 20° C. to 30° C.) for 5 minutes, followed by one wash in 2×SSC, 0.5% SDS at 57-58° C. for 20 minutes. An optional further wash can be conducted in 1×SSC, 0.5% SDS at 57-58° C. for 30 minutes, followed, if so desired, by an additional, optional, wash in 0.5×SSC, 0.5% SDS at 57-58° C. for 30 minutes.

In yet another approach, the entire Forsythia intermedia plr_Fi1 cDNA (SEQ ID NO:47) can be used as a probe to screen cDNA or genomic DNA libraries for full-length, or substantially full-length, pinoresinol/lariciresinol reductase clones. Representative hybridization conditions are 6×SSC, 5×Denhardt's, 0.5% SDS (Sigma) at 47° C. for 3 hours, followed by one wash at 6×SSC, 0.5% SDS (Sigma) at room temperature (typically 20° C. to 30° C.) for 3 minutes, then 4×SSC, 0.5% SDS (Sigma) at 47° C. for 2 minutes. An optional further wash can be conducted in 1×SSC, 0.5% SDS at 55° C. for 30 minutes, followed, if so desired, by an additional, optional, wash in 0.5×SSC, 0.5% SDS at 55° C. for 30 minutes.

Thus, in one aspect the present invention provides isolated nucleotide sequences encoding pinoresinol/lariciresinol reductase proteins, the nucleotide sequences being capable of remaining hybridized to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71, 107 and 117 under wash conditions of 1×SSC at 55° C. The present invention also provides vectors, such as replicable expression vectors, that include one or more nucleic acid molecules of the invention, and host cells that include one or more vectors of the invention.

The present invention also provides methods of enhancing the expression of pinoresinol/lariciresinol reductase protein in a host cell. The methods include the steps of (a) introducing into the host cell a replicable expression vector comprising a nucleic acid sequence encoding a pinoresinol/lariciresinol reductase protein, said nucleic acid sequence being capable of remaining hybridized to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71, 107 and 117, under stringent wash conditions, and (b) expressing the encoded pinoresinol/lariciresinol reductase protein.

The present invention also provides methods of inhibiting the expression of pinoresinol/lariciresinol reductase protein in a host cell. The methods include the steps of (a) introducing into the host cell a replicable vector that comprises a nucleic acid sequence capable of remaining hybridized to a nucleic acid sequence selected from the group consisting of SEQ ID NOS:47, 49, 51, 53, 55, 57, 61, 63, 65, 67, 69, 71, 107 and 117 under stringent wash conditions and (b) transcribing said nucleic acid sequence.

While the preferred embodiment of the invention has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the invention.

122 1 28 PRT Forsythia x intermedia PEPTIDE (1)..(28) Peptide fragment, wherein Xaa = unknown amino acid 1 Lys Pro Arg Pro Xaa Arg Xaa Xaa Lys Glu Leu Val Phe Tyr Phe Xaa 1 5 10 15 Asp Ile Leu Phe Lys Gly Xaa Asn Tyr Asn Xaa Ala 20 25 2 24 PRT Forsythia x intermedia 2 Thr Ala Met Ala Val Pro Phe Asn Tyr Gly Asp Leu Val Val Phe Asp 1 5 10 15 Asp Pro Ile Thr Leu Asp Asn Asn 20 3 16 PRT Forsythia x intermedia PEPTIDE (1)..(16) Peptide fragment, wherein Xaa = unknown amino acid 3 Tyr Val Gly Thr Leu Asn Phe Ala Gly Ala Asp Pro Leu Leu Xaa Lys 1 5 10 15 4 15 PRT Forsythia x intermedia 4 Asp Ile Ser Val Ile Gly Gly Thr Gly Asp Phe Phe Met Ala Arg 1 5 10 15 5 15 PRT Forsythia x intermedia PEPTIDE (1)..(15) Peptide fragment, wherein Xaa = unknown amino acid 5 Gly Val Ala Thr Leu Met Thr Asp Ala Phe Glu Gly Asp Xaa Tyr 1 5 10 15 6 10 PRT Forsythia x intermedia 6 Ala Gln Gly Met Tyr Phe Tyr Asp Gln Lys 1 5 10 7 5 PRT Forsythia x intermedia 7 Tyr Asn Ala Trp Leu 1 5 8 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 8 aargarytng tnttytaytt y 21 9 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 9 tarttraang gnacngccat 20 10 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 10 gtnatnggrt crtcraanac 20 11 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 11 ccatraaraa rtcnccngt 19 12 901 DNA Forsythia x intermedia CDS (26)..(583) 12 atttcggcac gagattaaac caaac atg gtt tct aaa aca caa att gta gct 52 Met Val Ser Lys Thr Gln Ile Val Ala 1 5 ctt ttc ctt tgc ttc ctc act tcc acc tct tcc gcc acc tac ggc cgc 100 Leu Phe Leu Cys Phe Leu Thr Ser Thr Ser Ser Ala Thr Tyr Gly Arg 10 15 20 25 aag cca cgc cct cgc cgg ccc tgc aaa gaa ttg gtg ttc tat ttc cac 148 Lys Pro Arg Pro Arg Arg Pro Cys Lys Glu Leu Val Phe Tyr Phe His 30 35 40 gac gta ctt ttc aaa gga aat aat tac cac aat gcc act tcc gcc ata 196 Asp Val Leu Phe Lys Gly Asn Asn Tyr His Asn Ala Thr Ser Ala Ile 45 50 55 gtc ggg tcc ccc caa tgg ggc aac aag act gcc atg gcc gtg cca ttc 244 Val Gly Ser Pro Gln Trp Gly Asn Lys Thr Ala Met Ala Val Pro Phe 60 65 70 aat tat ggt gac cta gtt gtg ttc gac gat ccc att acc tta gac aac 292 Asn Tyr Gly Asp Leu Val Val Phe Asp Asp Pro Ile Thr Leu Asp Asn 75 80 85 aat ctg cat tca ccc cca gtg ggt cgg gcg caa ggg atg tac ttc tat 340 Asn Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Met Tyr Phe Tyr 90 95 100 105 gat caa aaa aat aca tac aat gct tgg cta ggg ttc tca ttt ttg ttc 388 Asp Gln Lys Asn Thr Tyr Asn Ala Trp Leu Gly Phe Ser Phe Leu Phe 110 115 120 aat tca act aag tat gtt gga acc ttg aac ttt gct ggg gct gat cca 436 Asn Ser Thr Lys Tyr Val Gly Thr Leu Asn Phe Ala Gly Ala Asp Pro 125 130 135 ttg ttg aac aag act aga gac ata tca gtc att ggt gga act ggt gac 484 Leu Leu Asn Lys Thr Arg Asp Ile Ser Val Ile Gly Gly Thr Gly Asp 140 145 150 ttt ttc atg gcg aga ggg gtt gcc act ttg atg acc gat gcc ttt gaa 532 Phe Phe Met Ala Arg Gly Val Ala Thr Leu Met Thr Asp Ala Phe Glu 155 160 165 ggg gat gtg tat ttc cgc ctt cgt gtc gat att aat ttg tat gaa tgt 580 Gly Asp Val Tyr Phe Arg Leu Arg Val Asp Ile Asn Leu Tyr Glu Cys 170 175 180 185 tgg taaacaattt agccgtatat atatatatat atggctatac atatttcata 633 Trp gaatccagat ttgctgtttc aaatgtgtgt ttctttagtt gtgccaccaa taaaaaaatg 693 tacacattat ttaataaata taattattta atgtgttcat ttttgaagtt aaatttaagt 753 tgtatttatt tgattatgta taaattctct attagtaaaa tagtcaaagt gacacatatt 813 caagacgaca tatgtaactt tatttcatat cttcaacaag ttcaataatg tcatatatat 873 tgtactattg aaaaaaaaaa aaaaaaaa 901 13 186 PRT Forsythia x intermedia 13 Met Val Ser Lys Thr Gln Ile Val Ala Leu Phe Leu Cys Phe Leu Thr 1 5 10 15 Ser Thr Ser Ser Ala Thr Tyr Gly Arg Lys Pro Arg Pro Arg Arg Pro 20 25 30 Cys Lys Glu Leu Val Phe Tyr Phe His Asp Val Leu Phe Lys Gly Asn 35 40 45 Asn Tyr His Asn Ala Thr Ser Ala Ile Val Gly Ser Pro Gln Trp Gly 50 55 60 Asn Lys Thr Ala Met Ala Val Pro Phe Asn Tyr Gly Asp Leu Val Val 65 70 75 80 Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Pro Val 85 90 95 Gly Arg Ala Gln Gly Met Tyr Phe Tyr Asp Gln Lys Asn Thr Tyr Asn 100 105 110 Ala Trp Leu Gly Phe Ser Phe Leu Phe Asn Ser Thr Lys Tyr Val Gly 115 120 125 Thr Leu Asn Phe Ala Gly Ala Asp Pro Leu Leu Asn Lys Thr Arg Asp 130 135 140 Ile Ser Val Ile Gly Gly Thr Gly Asp Phe Phe Met Ala Arg Gly Val 145 150 155 160 Ala Thr Leu Met Thr Asp Ala Phe Glu Gly Asp Val Tyr Phe Arg Leu 165 170 175 Arg Val Asp Ile Asn Leu Tyr Glu Cys Trp 180 185 14 858 DNA Forsythia x intermedia CDS (19)..(573) 14 aattcggcac gaggaaaa atg gca gct aaa aca caa acc aca gcc ctt ttc 51 Met Ala Ala Lys Thr Gln Thr Thr Ala Leu Phe 1 5 10 ctc tgc ctc ctc atc tgc atc tcc gcc gtg tac ggc cac aaa acc agg 99 Leu Cys Leu Leu Ile Cys Ile Ser Ala Val Tyr Gly His Lys Thr Arg 15 20 25 tct cga cgc ccc tgt aaa gag ctc gtt ttc ttc ttc cac gac atc ctc 147 Ser Arg Arg Pro Cys Lys Glu Leu Val Phe Phe Phe His Asp Ile Leu 30 35 40 tac cta gga tac aat aga aac aat gcc acc gct gtc ata gta gcc tct 195 Tyr Leu Gly Tyr Asn Arg Asn Asn Ala Thr Ala Val Ile Val Ala Ser 45 50 55 cct caa tgg gga aac aag act gcc atg gct aaa cct ttc aat ttt ggt 243 Pro Gln Trp Gly Asn Lys Thr Ala Met Ala Lys Pro Phe Asn Phe Gly 60 65 70 75 gat ttg gtt gtg ttt gat gat ccc att acc tta gac aac aac ctg cat 291 Asp Leu Val Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His 80 85 90 tct cct ccg gtc ggc cgg gct cag gga act tat ttc tac gat caa tgg 339 Ser Pro Pro Val Gly Arg Ala Gln Gly Thr Tyr Phe Tyr Asp Gln Trp 95 100 105 agt att tat ggt gca tgg ctt gga ttt tca ttt ttg ttc aat tct act 387 Ser Ile Tyr Gly Ala Trp Leu Gly Phe Ser Phe Leu Phe Asn Ser Thr 110 115 120 gat tat gtt gga act cta aat ttt gct gga gct gat cca ttg att aac 435 Asp Tyr Val Gly Thr Leu Asn Phe Ala Gly Ala Asp Pro Leu Ile Asn 125 130 135 aaa act agg gac att tca gta att gga gga act ggt gat ttt ttc atg 483 Lys Thr Arg Asp Ile Ser Val Ile Gly Gly Thr Gly Asp Phe Phe Met 140 145 150 155 gct aga ggg gta gcc act gtg tcg acc gat gct ttt gaa ggg gat gtt 531 Ala Arg Gly Val Ala Thr Val Ser Thr Asp Ala Phe Glu Gly Asp Val 160 165 170 tat ttc agg ctt cgt gtt gat att agg ttg tat gag tgt tgg 573 Tyr Phe Arg Leu Arg Val Asp Ile Arg Leu Tyr Glu Cys Trp 175 180 185 taaatttacc ttatttttcc attttcttga gtttgactcg gatttgacta ataatgtctt 633 ctgtaatcct tgtttttgat caatttgtgg cgattttatc aattagtgat tgtttggttc 693 atattttaat ctgttaaaaa aaattgtggt caaaagccaa taaccacaac cgtagggagt 753 tttttccgtt aaggggaaaa aaaagtatgt ccatgtgtta ctacgttttc aatttcattc 813 aaaatttgct tttcaatcat cttcttcaaa aaaaaaaaaa aaaaa 858 15 185 PRT Forsythia x intermedia 15 Met Ala Ala Lys Thr Gln Thr Thr Ala Leu Phe Leu Cys Leu Leu Ile 1 5 10 15 Cys Ile Ser Ala Val Tyr Gly His Lys Thr Arg Ser Arg Arg Pro Cys 20 25 30 Lys Glu Leu Val Phe Phe Phe His Asp Ile Leu Tyr Leu Gly Tyr Asn 35 40 45 Arg Asn Asn Ala Thr Ala Val Ile Val Ala Ser Pro Gln Trp Gly Asn 50 55 60 Lys Thr Ala Met Ala Lys Pro Phe Asn Phe Gly Asp Leu Val Val Phe 65 70 75 80 Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Pro Val Gly 85 90 95 Arg Ala Gln Gly Thr Tyr Phe Tyr Asp Gln Trp Ser Ile Tyr Gly Ala 100 105 110 Trp Leu Gly Phe Ser Phe Leu Phe Asn Ser Thr Asp Tyr Val Gly Thr 115 120 125 Leu Asn Phe Ala Gly Ala Asp Pro Leu Ile Asn Lys Thr Arg Asp Ile 130 135 140 Ser Val Ile Gly Gly Thr Gly Asp Phe Phe Met Ala Arg Gly Val Ala 145 150 155 160 Thr Val Ser Thr Asp Ala Phe Glu Gly Asp Val Tyr Phe Arg Leu Arg 165 170 175 Val Asp Ile Arg Leu Tyr Glu Cys Trp 180 185 16 948 DNA Tsuga heterophylla CDS (104)..(688) 16 gggcaccctc tcttgttaat tgagcccttc tcctcctact tctcttgtta gttctttgat 60 cccatatctt cttctataat cactttagtc tataagattg tca atg gca atc aag 115 Met Ala Ile Lys 1 aat cgt aat aga gct gtg cac ttg tgt ttt cta tgg ctt cta ctg tcc 163 Asn Arg Asn Arg Ala Val His Leu Cys Phe Leu Trp Leu Leu Leu Ser 5 10 15 20 tct gtg ttg ttg caa aca agt gat ggg aaa agc tgg aag aag cac cga 211 Ser Val Leu Leu Gln Thr Ser Asp Gly Lys Ser Trp Lys Lys His Arg 25 30 35 ctc cga aag cct tgt agg aat ctg gtg ttg tat ttc cat gat gta atc 259 Leu Arg Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp Val Ile 40 45 50 tac aat ggc agc aac gcc aag aac gct aca tcc aca ctt gtg ggt gct 307 Tyr Asn Gly Ser Asn Ala Lys Asn Ala Thr Ser Thr Leu Val Gly Ala 55 60 65 ccc cac ggg tct aac ctc aca ctt ctc gct gga aaa gac aac cac ttt 355 Pro His Gly Ser Asn Leu Thr Leu Leu Ala Gly Lys Asp Asn His Phe 70 75 80 gga gat ctg gcg gtg ttt gac gat ccc atc act ctt gac aac aat ttc 403 Gly Asp Leu Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Phe 85 90 95 100 cac tct cct ccg gtg ggc aga gct cag gga ttc tac ttt tat gac atg 451 His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met 105 110 115 aag aac acc ttc agc tcc tgg ctt gga ttc acg ttt gta ctc aac tct 499 Lys Asn Thr Phe Ser Ser Trp Leu Gly Phe Thr Phe Val Leu Asn Ser 120 125 130 aca gat tac aaa ggc acc atc acg ttc tct gga gcc gat cca atc ctt 547 Thr Asp Tyr Lys Gly Thr Ile Thr Phe Ser Gly Ala Asp Pro Ile Leu 135 140 145 act aaa tac aga gat ata tca gtg gtg gga gga act gga gat ttc ata 595 Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Ile 150 155 160 atg gca aga gga atc gcc aca atc tcc acc gat gcg tat gaa ggc gac 643 Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ala Tyr Glu Gly Asp 165 170 175 180 gtt tac ttc cgt ctc tgc gtg aat atc aca ctc tat gag tgc tac 688 Val Tyr Phe Arg Leu Cys Val Asn Ile Thr Leu Tyr Glu Cys Tyr 185 190 195 tgagtgctat aggtctattt tctccttcga ctatccattt atatgttcat tttagttgaa 748 ctagtgtttt cttgtgcgag agatatgcac gaagctctga gatattgtag cgtgaagttc 808 ctttagcagc cgaataatgt atttcgattt tgtcgaaggc catatctaat attgtcaagg 868 gaaaatgcag aattctatgt cggtcaagca cttttattta aaaataaaag aaatattggt 928 taaaaaaaaa aaaaaaaaaa 948 17 195 PRT Tsuga heterophylla 17 Met Ala Ile Lys Asn Arg Asn Arg Ala Val His Leu Cys Phe Leu Trp 1 5 10 15 Leu Leu Leu Ser Ser Val Leu Leu Gln Thr Ser Asp Gly Lys Ser Trp 20 25 30 Lys Lys His Arg Leu Arg Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe 35 40 45 His Asp Val Ile Tyr Asn Gly Ser Asn Ala Lys Asn Ala Thr Ser Thr 50 55 60 Leu Val Gly Ala Pro His Gly Ser Asn Leu Thr Leu Leu Ala Gly Lys 65 70 75 80 Asp Asn His Phe Gly Asp Leu Ala Val Phe Asp Asp Pro Ile Thr Leu 85 90 95 Asp Asn Asn Phe His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr 100 105 110 Phe Tyr Asp Met Lys Asn Thr Phe Ser Ser Trp Leu Gly Phe Thr Phe 115 120 125 Val Leu Asn Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Ser Gly Ala 130 135 140 Asp Pro Ile Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr 145 150 155 160 Gly Asp Phe Ile Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ala 165 170 175 Tyr Glu Gly Asp Val Tyr Phe Arg Leu Cys Val Asn Ile Thr Leu Tyr 180 185 190 Glu Cys Tyr 195 18 849 DNA Tsuga heterophylla CDS (71)..(625) 18 gttctgttcc aaattctaat tagccttcca ttcattccag gatcccactc ttcttccttc 60 aagattggca atg gct atc aag agt aat agg gct gtg cgt ttc tgc ttt 109 Met Ala Ile Lys Ser Asn Arg Ala Val Arg Phe Cys Phe 1 5 10 gta tgg ctt ctg ttg ttg caa agt ggt ttt gta ttt cca ctc cca cag 157 Val Trp Leu Leu Leu Leu Gln Ser Gly Phe Val Phe Pro Leu Pro Gln 15 20 25 cct tgt agg aat ctg gtt ttg tat ttc cac gat gta ctc tac aat ggc 205 Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp Val Leu Tyr Asn Gly 30 35 40 45 ttc aac gcc cac aac gct aca tct aca ctt gtg ggt gct cca cag ggg 253 Phe Asn Ala His Asn Ala Thr Ser Thr Leu Val Gly Ala Pro Gln Gly 50 55 60 gct aac ctc aca ctt ctc gct gga aaa gac aac cac ttt gga gat ctg 301 Ala Asn Leu Thr Leu Leu Ala Gly Lys Asp Asn His Phe Gly Asp Leu 65 70 75 gcg gtg ttc gac gat ccc atc act ctt gac aac aat ttc cag tct cct 349 Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Phe Gln Ser Pro 80 85 90 ccg gtg ggc aga gct cag gga ttc tac ttt tat gac atg aag aac acc 397 Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asn Thr 95 100 105 ttc agc tcc tgg ctt gga ttc acg ttt gta ctc aac tct aca gat tac 445 Phe Ser Ser Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp Tyr 110 115 120 125 aaa ggc acc atc acg ttc tct gga gcc gat cca atc ctt act aaa tac 493 Lys Gly Thr Ile Thr Phe Ser Gly Ala Asp Pro Ile Leu Thr Lys Tyr 130 135 140 aga gat ata tca gtg gtg gga gga act gga gat ttc ata atg gca aga 541 Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Ile Met Ala Arg 145 150 155 gga atc gcc aca atc tcc acc gat gcg tat gaa gga gat gtt tac ttc 589 Gly Ile Ala Thr Ile Ser Thr Asp Ala Tyr Glu Gly Asp Val Tyr Phe 160 165 170 cgt ctc cgc gtc aat atc aca ctc tat gaa tgc tac tgatattatt 635 Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 175 180 185 aagtagctac tgtttctcgt ctggtctcgc catttcgatg ctctttttaa cattagtgct 695 ttccataaat tgttgtagcc tctcaataaa acccagtaaa atatttcttc tgtttattta 755 gcagcttcca aatcattgta ttagtatttt atattatttg gattttatac aagtccataa 815 aatatttctt cagctaaaaa aaaaaaaaaa aaaa 849 19 185 PRT Tsuga heterophylla 19 Met Ala Ile Lys Ser Asn Arg Ala Val Arg Phe Cys Phe Val Trp Leu 1 5 10 15 Leu Leu Leu Gln Ser Gly Phe Val Phe Pro Leu Pro Gln Pro Cys Arg 20 25 30 Asn Leu Val Leu Tyr Phe His Asp Val Leu Tyr Asn Gly Phe Asn Ala 35 40 45 His Asn Ala Thr Ser Thr Leu Val Gly Ala Pro Gln Gly Ala Asn Leu 50 55 60 Thr Leu Leu Ala Gly Lys Asp Asn His Phe Gly Asp Leu Ala Val Phe 65 70 75 80 Asp Asp Pro Ile Thr Leu Asp Asn Asn Phe Gln Ser Pro Pro Val Gly 85 90 95 Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asn Thr Phe Ser Ser 100 105 110 Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp Tyr Lys Gly Thr 115 120 125 Ile Thr Phe Ser Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg Asp Ile 130 135 140 Ser Val Val Gly Gly Thr Gly Asp Phe Ile Met Ala Arg Gly Ile Ala 145 150 155 160 Thr Ile Ser Thr Asp Ala Tyr Glu Gly Asp Val Tyr Phe Arg Leu Arg 165 170 175 Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 20 873 DNA Thuja plicata CDS (25)..(591) 20 gttgcacgag ggatttcaag agat atg agt aga ata gca ttt cat ttg tgc 51 Met Ser Arg Ile Ala Phe His Leu Cys 1 5 ttc atg ggg ctt ctg ctc tct tcc acg gtg ctc aga aat gta gat ggg 99 Phe Met Gly Leu Leu Leu Ser Ser Thr Val Leu Arg Asn Val Asp Gly 10 15 20 25 cat gca tgg aag agg caa ctt cca atg cca tgt aag aat ttg gtg ctc 147 His Ala Trp Lys Arg Gln Leu Pro Met Pro Cys Lys Asn Leu Val Leu 30 35 40 tac ttt cat gat ata ctc tac aat ggc aaa aac att cac aat gca act 195 Tyr Phe His Asp Ile Leu Tyr Asn Gly Lys Asn Ile His Asn Ala Thr 45 50 55 gct gcg ctg gtt gca gct cct gcg tgg ggc aat ctc act act ttc gct 243 Ala Ala Leu Val Ala Ala Pro Ala Trp Gly Asn Leu Thr Thr Phe Ala 60 65 70 gaa cct ttc aag ttt gga gat gtg gtt gtg ttt gac gat ccc att act 291 Glu Pro Phe Lys Phe Gly Asp Val Val Val Phe Asp Asp Pro Ile Thr 75 80 85 ctc gac aac aat ctt cac tct cct cct gtg gga aga gcg cag gga ttt 339 Leu Asp Asn Asn Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe 90 95 100 105 tat ttg tac aac atg aag act act tac aat gct tgg ttg ggg ttc aca 387 Tyr Leu Tyr Asn Met Lys Thr Thr Tyr Asn Ala Trp Leu Gly Phe Thr 110 115 120 ttt gtg ctg aat tcg aca gat tat aag ggc aca atc acc ttc aat ggc 435 Phe Val Leu Asn Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Asn Gly 125 130 135 gcc gac ccc ccg ctg gtt aag tac aga gat ata tcc gtt gtt ggc ggt 483 Ala Asp Pro Pro Leu Val Lys Tyr Arg Asp Ile Ser Val Val Gly Gly 140 145 150 acg ggt gat ttc ttg atg gcg aga gga att gcc acc ctt tct act gat 531 Thr Gly Asp Phe Leu Met Ala Arg Gly Ile Ala Thr Leu Ser Thr Asp 155 160 165 gca atc gag gga aat gtt tat ttc cga ctc agg gtt aac atc aca ctc 579 Ala Ile Glu Gly Asn Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu 170 175 180 185 tac gag tgt tac tgatgattac taactaaatg gagagtcttt gtttagagaa 631 Tyr Glu Cys Tyr tagtgtgttg ggctgtttac ttaaagtcga cgttctatgc agttgaagtc tttgtttaga 691 tgaatgcaat ggtgggtttt ctttcctcgt gagggttaac atcacactct acgagtgtta 751 ctgataattg ttaagtattt ggagagtctt gtaagttgag aataatgtat tttggctgtt 811 tattttgagt cgaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa 871 aa 873 21 189 PRT Thuja plicata 21 Met Ser Arg Ile Ala Phe His Leu Cys Phe Met Gly Leu Leu Leu Ser 1 5 10 15 Ser Thr Val Leu Arg Asn Val Asp Gly His Ala Trp Lys Arg Gln Leu 20 25 30 Pro Met Pro Cys Lys Asn Leu Val Leu Tyr Phe His Asp Ile Leu Tyr 35 40 45 Asn Gly Lys Asn Ile His Asn Ala Thr Ala Ala Leu Val Ala Ala Pro 50 55 60 Ala Trp Gly Asn Leu Thr Thr Phe Ala Glu Pro Phe Lys Phe Gly Asp 65 70 75 80 Val Val Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser 85 90 95 Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Leu Tyr Asn Met Lys Thr 100 105 110 Thr Tyr Asn Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp 115 120 125 Tyr Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Pro Leu Val Lys 130 135 140 Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala 145 150 155 160 Arg Gly Ile Ala Thr Leu Ser Thr Asp Ala Ile Glu Gly Asn Val Tyr 165 170 175 Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 22 867 DNA Thuja plicata CDS (80)..(655) 22 gcaatattgt gctggttcag taatctatgt cttgttgacc tgtagtgtat acccaaacat 60 ttctccttct tttgcaaaa atg gca atg aag gct gca aaa ttt ctg cat ttc 112 Met Ala Met Lys Ala Ala Lys Phe Leu His Phe 1 5 10 tta ttt atc tgg ctt cta gtc tgc act gtg ttg ctc aaa tct gca gac 160 Leu Phe Ile Trp Leu Leu Val Cys Thr Val Leu Leu Lys Ser Ala Asp 15 20 25 tgt cat aga tgg aag aag aaa att cca gag cca tgt aag aat ctg gta 208 Cys His Arg Trp Lys Lys Lys Ile Pro Glu Pro Cys Lys Asn Leu Val 30 35 40 ttg tac ttt cat gat atc ctc tac aat gga tcc aac aaa cac aat gca 256 Leu Tyr Phe His Asp Ile Leu Tyr Asn Gly Ser Asn Lys His Asn Ala 45 50 55 aca tct gca att gtt gga gca ccc aaa gga gcc aat ctc act att ttg 304 Thr Ser Ala Ile Val Gly Ala Pro Lys Gly Ala Asn Leu Thr Ile Leu 60 65 70 75 act ggt aac aac cat ttt gga gat gtg gtt gtg ttt gat gat cct att 352 Thr Gly Asn Asn His Phe Gly Asp Val Val Val Phe Asp Asp Pro Ile 80 85 90 act ctt gac aac aat ctt cac tct act cct gtg gga aga gct cag ggc 400 Thr Leu Asp Asn Asn Leu His Ser Thr Pro Val Gly Arg Ala Gln Gly 95 100 105 ttt tat ttc tat gac atg aag aat aca ttc aat tct tgg ctt ggg ttt 448 Phe Tyr Phe Tyr Asp Met Lys Asn Thr Phe Asn Ser Trp Leu Gly Phe 110 115 120 aca ttt gtg ttg aat tca aca aat tat aag ggc acc atc acc ttc aat 496 Thr Phe Val Leu Asn Ser Thr Asn Tyr Lys Gly Thr Ile Thr Phe Asn 125 130 135 ggg gct gac cca att ctg act aag tac aga gat ata tct gtt gtg ggt 544 Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly 140 145 150 155 ggt acg ggt gat ttc ttg atg gcc aga gga atc gcc acc att tct act 592 Gly Thr Gly Asp Phe Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr 160 165 170 gat gca tac gag gga gat gtt tat ttc cgt ctt agg gtg aat atc act 640 Asp Ala Tyr Glu Gly Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr 175 180 185 ctc tat gag tgt tac tgattcgaat ttgatttcct gttctaatct ctaatttgag 695 Leu Tyr Glu Cys Tyr 190 aggatgaaca ttcaataaac tttatagaag catatataaa taggtgcagg aaaataagag 755 gtaagggatg agattatttc agcctcatat cttattctgc atcagttttg tatgctcatt 815 tgtttaataa aatttgacca gtttcatcat gttgaaaaaa aaaaaaaaaa aa 867 23 192 PRT Thuja plicata 23 Met Ala Met Lys Ala Ala Lys Phe Leu His Phe Leu Phe Ile Trp Leu 1 5 10 15 Leu Val Cys Thr Val Leu Leu Lys Ser Ala Asp Cys His Arg Trp Lys 20 25 30 Lys Lys Ile Pro Glu Pro Cys Lys Asn Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Leu Tyr Asn Gly Ser Asn Lys His Asn Ala Thr Ser Ala Ile Val 50 55 60 Gly Ala Pro Lys Gly Ala Asn Leu Thr Ile Leu Thr Gly Asn Asn His 65 70 75 80 Phe Gly Asp Val Val Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 Leu His Ser Thr Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asn Thr Phe Asn Ser Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asn Tyr Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ala Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 24 914 DNA Thuja plicata CDS (94)..(669) 24 cgtaggaaat atctcagagg gagccgaaaa ttgagataat tgttgtacga aatatataaa 60 agattagatt cagaggaatt tgcagatgtt gtt gta tct aaa aca gct gct aga 114 Val Ser Lys Thr Ala Ala Arg 1 5 gtt ctg cat tta tgc ttt cta tgg ctt cta gta tct gca atc ttc ata 162 Val Leu His Leu Cys Phe Leu Trp Leu Leu Val Ser Ala Ile Phe Ile 10 15 20 aaa tct gca gat tgc cgt agc tgg aaa aag aag ctt cca aag ccc tgt 210 Lys Ser Ala Asp Cys Arg Ser Trp Lys Lys Lys Leu Pro Lys Pro Cys 25 30 35 aga aat ctt gtg tta tat ttt cat gat ata atc tac aat ggc aaa aat 258 Arg Asn Leu Val Leu Tyr Phe His Asp Ile Ile Tyr Asn Gly Lys Asn 40 45 50 55 gca gag aat gca aca tct gca ctt gtt tca gcc cct caa gga gct aat 306 Ala Glu Asn Ala Thr Ser Ala Leu Val Ser Ala Pro Gln Gly Ala Asn 60 65 70 ctc acc att atg act ggt aat aac cat ttt ggg aat ctt gca gtg ttt 354 Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly Asn Leu Ala Val Phe 75 80 85 gat gat cct att act ctt gac aac aat ctt cac tct cct cct gtt gga 402 Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Pro Val Gly 90 95 100 aga gct cag ggc ttt tac ttc tat gac atg aag aac acc ttc agt gcc 450 Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asn Thr Phe Ser Ala 105 110 115 tgg ctt ggc ttc aca ttt gtg ctc aat tca act gat cac aag ggc tcc 498 Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp His Lys Gly Ser 120 125 130 135 att act ttc aat gga gca gat ccc atc tta aca aag tac aga gac ata 546 Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg Asp Ile 140 145 150 tct gtt gtg ggt gga aca ggg gat ttc ttg atg gca aga gga att gct 594 Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala Arg Gly Ile Ala 155 160 165 acc att tct act gac tca tat gag gga gat gtt tat ttc agg ctt agg 642 Thr Ile Ser Thr Asp Ser Tyr Glu Gly Asp Val Tyr Phe Arg Leu Arg 170 175 180 gtc aat atc aca ctc tat gag tgt tac tgaacaaatt ccttgctctg 689 Val Asn Ile Thr Leu Tyr Glu Cys Tyr 185 190 tatttctagt ttttgggacc ttttaaagat agttgtttac ttcaatgtct ctatatgtaa 749 taacactgtg tgaagattat atacgatgga ctatagaaac tatgttgaat tctgttctgt 809 agctaattta tgtatatgat ccactcatat ctcttaatat gataccgatt tgtaattatc 869 ccagataaag tatgtcatgt gctttgacaa aaaaaaaaaa aaaaa 914 25 192 PRT Thuja plicata 25 Val Ser Lys Thr Ala Ala Arg Val Leu His Leu Cys Phe Leu Trp Leu 1 5 10 15 Leu Val Ser Ala Ile Phe Ile Lys Ser Ala Asp Cys Arg Ser Trp Lys 20 25 30 Lys Lys Leu Pro Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Ile Tyr Asn Gly Lys Asn Ala Glu Asn Ala Thr Ser Ala Leu Val 50 55 60 Ser Ala Pro Gln Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His 65 70 75 80 Phe Gly Asn Leu Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asp His Lys Gly Ser Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 26 704 DNA Thuja plicata CDS (3)..(416) 26 ag aat gcc cac aat gca aca tct gca ctt gtt gca gcc cct gag gga 47 Asn Ala His Asn Ala Thr Ser Ala Leu Val Ala Ala Pro Glu Gly 1 5 10 15 gcc aat ctc acc att atg act ggt aat aac cat ttt ggg aat att gct 95 Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly Asn Ile Ala 20 25 30 gtg ttt gat gat cct att act ctt gac aac aat ctt cac tct cct tct 143 Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Ser 35 40 45 gtt gga aga gct cag ggc ttt tac ttc tat gac atg aag gat acc ttc 191 Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asp Thr Phe 50 55 60 aat gct tgg ctt ggt ttt aca ttt gtg ctg aat tca act gat cac aag 239 Asn Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp His Lys 65 70 75 ggc acc att act ttc aat gga gca gat cca atc ctg acc aag tac aga 287 Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg 80 85 90 95 gat ata tct gtt gtg ggt gga aca ggg gat ttc ttg atg gcc aga gga 335 Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala Arg Gly 100 105 110 att gcc acc att tct act gat tca tat gag gga gat gtt tat ttc agg 383 Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly Asp Val Tyr Phe Arg 115 120 125 ctt agg gtc aat atc aca ctc tat gag tgt tac taaaaatgaa tttcctctgt 436 Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 130 135 attactagct tataggagtc attccctggt tcaatgtcta gggcatggaa taaaagaatt 496 tgaagatggt tttgaaatat ggagcatgta ttctaatttg aagagccctc aaggaagtgc 556 attttacaga gtttagtttt gccctctaga atattatgtt ttcaaaatgc tctatgaaag 616 tcatatgatg tatggagtac catttggaat aattaaagca agcatatttt attaaaaaaa 676 aaaaaaaaaa aaaaaaaaaa aaaaaaaa 704 27 138 PRT Thuja plicata 27 Asn Ala His Asn Ala Thr Ser Ala Leu Val Ala Ala Pro Glu Gly Ala 1 5 10 15 Asn Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly Asn Ile Ala Val 20 25 30 Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Ser Val 35 40 45 Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asp Thr Phe Asn 50 55 60 Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp His Lys Gly 65 70 75 80 Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg Asp 85 90 95 Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala Arg Gly Ile 100 105 110 Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly Asp Val Tyr Phe Arg Leu 115 120 125 Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 130 135 28 820 DNA Thuja plicata CDS (43)..(612) 28 gtctaattga gagaaaattc caataatttt ttaccaatag ca atg aaa gcc att 54 Met Lys Ala Ile 1 aga gtt ctg cat tta tgc ttt cta tgt ctt cta gtg tct gca atc ttg 102 Arg Val Leu His Leu Cys Phe Leu Cys Leu Leu Val Ser Ala Ile Leu 5 10 15 20 cta aaa tct gca gat tgc cat agc tgg aaa aag aag ctt cca aag ccc 150 Leu Lys Ser Ala Asp Cys His Ser Trp Lys Lys Lys Leu Pro Lys Pro 25 30 35 tgc aag aat ctt gtg tta tat ttc cat gat ata atc tac aat ggc aaa 198 Cys Lys Asn Leu Val Leu Tyr Phe His Asp Ile Ile Tyr Asn Gly Lys 40 45 50 aat gca gag aat gca aca tct gca ctt gtt gca gcc cct gag gga gcc 246 Asn Ala Glu Asn Ala Thr Ser Ala Leu Val Ala Ala Pro Glu Gly Ala 55 60 65 aat ctc acc att atg act ggt aat aac cat ttt ggg aat ctt gct gtg 294 Asn Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly Asn Leu Ala Val 70 75 80 ttt gat gat cct att act ctt gac aac aat ctc cac tct cct cct gtg 342 Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Pro Val 85 90 95 100 gga aga gct cag gga ttt tac ttc tat gac atg aag aac acc ttc agt 390 Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asn Thr Phe Ser 105 110 115 gct tgg ctt ggc ttc aca ttt gtg ctg aat tca act gat cac aag ggc 438 Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp His Lys Gly 120 125 130 acc att act ttc aat gga gca gac cca atc ctg acc aag tac aga gac 486 Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg Asp 135 140 145 ata tct gtt gtg ggt gga aca ggg gat ttc ttg atg gcc aga gga att 534 Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala Arg Gly Ile 150 155 160 gcc acc att tct act gat tca tat gag gga gaa gtt tat ttc agg ctt 582 Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly Glu Val Tyr Phe Arg Leu 165 170 175 180 agg gtc aat atc aca ctc tat gag tgt tac tgagcaaatg cctgtcttct 632 Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 185 190 tcctctgtag ttcttgtttt gggtgccttt gaggaatagt tcttggcttc aatgtctctg 692 tatgtagtaa catggtcaat ggagtctatt ttgaagatta tgaagatata gtctctatat 752 atatatatat tgaagagaat gagatctgtt ttaggtagct cttttcattc aaaaaaaaaa 812 aaaaaaaa 820 29 190 PRT Thuja plicata 29 Met Lys Ala Ile Arg Val Leu His Leu Cys Phe Leu Cys Leu Leu Val 1 5 10 15 Ser Ala Ile Leu Leu Lys Ser Ala Asp Cys His Ser Trp Lys Lys Lys 20 25 30 Leu Pro Lys Pro Cys Lys Asn Leu Val Leu Tyr Phe His Asp Ile Ile 35 40 45 Tyr Asn Gly Lys Asn Ala Glu Asn Ala Thr Ser Ala Leu Val Ala Ala 50 55 60 Pro Glu Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly 65 70 75 80 Asn Leu Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His 85 90 95 Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys 100 105 110 Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr 115 120 125 Asp His Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr 130 135 140 Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met 145 150 155 160 Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly Glu Val 165 170 175 Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 30 1013 DNA Thuja plicata CDS (47)..(616) 30 ctcagtctaa ttgagagaaa attccaataa ttttttccca atagca atg aaa gcc 55 Met Lys Ala 1 att aga gtt ctg caa tta tgc ttt cta tgg ctt cta gta tct gca atc 103 Ile Arg Val Leu Gln Leu Cys Phe Leu Trp Leu Leu Val Ser Ala Ile 5 10 15 ttg cta aaa tct gca gat tgc cat agc tgg aaa aag aag ctt cca aag 151 Leu Leu Lys Ser Ala Asp Cys His Ser Trp Lys Lys Lys Leu Pro Lys 20 25 30 35 ccc tgc aag aat ctt gtg tta tat ttc cat gat ata atc tac aat ggc 199 Pro Cys Lys Asn Leu Val Leu Tyr Phe His Asp Ile Ile Tyr Asn Gly 40 45 50 aaa aat gca gag aat gca aca tct gca ctt gtt gca gcc cct gag gga 247 Lys Asn Ala Glu Asn Ala Thr Ser Ala Leu Val Ala Ala Pro Glu Gly 55 60 65 gcc aat ctc acc att atg act ggt aat aac cat ttt ggg aat ctt gct 295 Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly Asn Leu Ala 70 75 80 gtg ttt gat gat cct att act ctt gac aac aat ctc cac tct cct cct 343 Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His Ser Pro Pro 85 90 95 gtg gga aga gct cag ggc ttt tac ttc tat gac atg aag aac acc ttc 391 Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asn Thr Phe 100 105 110 115 agt gct tgg ctt ggc ttc aca ttt gtg ctg aat tca act gat cac aag 439 Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp His Lys 120 125 130 ggc acc att act ttc aat gga gca gac cca atc ctg acc aag tac aga 487 Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr Lys Tyr Arg 135 140 145 gat ata tct gtt gtg ggt gga aca ggg gat ttc ttg atg gcc aga gga 535 Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala Arg Gly 150 155 160 att gcc acc att tct act gat tca tat gag gga gat gtt tat ttc agg 583 Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly Asp Val Tyr Phe Arg 165 170 175 ctt agg gtc aat atc aca ctc tat aag tgt tac tgagcaaatg cctgtcttct 636 Leu Arg Val Asn Ile Thr Leu Tyr Lys Cys Tyr 180 185 190 tcctctgtag ttcttgtttt gggtgccttt gaggaatagt tcttggcttc aatgtctctg 696 tatgtagtaa catggtcaat ggagtctatt ttgaagatta tgaagatata gtctctctat 756 atatatatat tgaagagaat gagatctgtt ttaggtagct cttttcattc atatatatgg 816 gttaacttgg atttcatgtt tggttcaaag atcagttatg gaggatttcc ttttagtggt 876 tttatgggat ttttgacata ttagattact ttcatctcaa atatatgtta aatcagttat 936 atatgaaact aatcatatat aagttcagaa atatcagaac aaccatttta tggaaaaaaa 996 aaaaaaaaaa aaaaaaa 1013 31 190 PRT Thuja plicata 31 Met Lys Ala Ile Arg Val Leu Gln Leu Cys Phe Leu Trp Leu Leu Val 1 5 10 15 Ser Ala Ile Leu Leu Lys Ser Ala Asp Cys His Ser Trp Lys Lys Lys 20 25 30 Leu Pro Lys Pro Cys Lys Asn Leu Val Leu Tyr Phe His Asp Ile Ile 35 40 45 Tyr Asn Gly Lys Asn Ala Glu Asn Ala Thr Ser Ala Leu Val Ala Ala 50 55 60 Pro Glu Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His Phe Gly 65 70 75 80 Asn Leu Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn Leu His 85 90 95 Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys 100 105 110 Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr 115 120 125 Asp His Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile Leu Thr 130 135 140 Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met 145 150 155 160 Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly Asp Val 165 170 175 Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Lys Cys Tyr 180 185 190 32 913 DNA Thuja plicata CDS (77)..(652) 32 gcaagctcaa atacccgact tctttctcta cttcagagct cttctttctt caaacatttt 60 tgatatattt tgcaca atg gca atc tgg aat gga aga gtt ctg aat ttg tgc 112 Met Ala Ile Trp Asn Gly Arg Val Leu Asn Leu Cys 1 5 10 att ctg tgg ctt ctg gtc tcc ata gtt ttg ctg aat ggt ata gat tgc 160 Ile Leu Trp Leu Leu Val Ser Ile Val Leu Leu Asn Gly Ile Asp Cys 15 20 25 cat agt aga aaa aag aag ctt cca aag cca tgt agg aat ctt gtt ttg 208 His Ser Arg Lys Lys Lys Leu Pro Lys Pro Cys Arg Asn Leu Val Leu 30 35 40 tat ttt cat gat att atc tac aat ggt aaa aat gca ggc aat gca aca 256 Tyr Phe His Asp Ile Ile Tyr Asn Gly Lys Asn Ala Gly Asn Ala Thr 45 50 55 60 tct acg ctt gtt gca gcc cct caa gga gct aat ctc acc att atg act 304 Ser Thr Leu Val Ala Ala Pro Gln Gly Ala Asn Leu Thr Ile Met Thr 65 70 75 ggc aat tac cat ttt gga gat ctg tct gtg ttt gat gat cct att act 352 Gly Asn Tyr His Phe Gly Asp Leu Ser Val Phe Asp Asp Pro Ile Thr 80 85 90 gtt gac aac aat ctt cat tct cct cct gtg gga aga gct cag ggc ttt 400 Val Asp Asn Asn Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe 95 100 105 tac ttc tat gac atg aag aat aca ttc agt gct tgg ctt ggg ttc aca 448 Tyr Phe Tyr Asp Met Lys Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr 110 115 120 ttt gtg ctg aac tca aca gat tat aaa ggc act att act ttc ggt gga 496 Phe Val Leu Asn Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Gly Gly 125 130 135 140 gca gac cca att ttg gct aag tac aga gat ata tct gtt gtg ggt ggt 544 Ala Asp Pro Ile Leu Ala Lys Tyr Arg Asp Ile Ser Val Val Gly Gly 145 150 155 act gga gat ttc ttg atg gca aga gga att gct aca atc gat act gat 592 Thr Gly Asp Phe Leu Met Ala Arg Gly Ile Ala Thr Ile Asp Thr Asp 160 165 170 gca tat gag gga gat gtt tat ttc agg cta agg gtg aat atc aca ctc 640 Ala Tyr Glu Gly Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu 175 180 185 tat gag tgt tac tgatccatgg gtattctatg tagaatagct caatctgata 692 Tyr Glu Cys Tyr 190 tggctatatt attttgagag cataggtagt taagttttat aactaagtag tgaaccatga 752 gatcattgaa aacttgggtg ctcatgcaca gttttcatat tttctaaata agtctgctcg 812 actattacat ttatggattg ttgagaattg tgtcgcttat tactttatga ataagctatt 872 ttaaacaaag ttttcacaag tttaaaaaaa aaaaaaaaaa a 913 33 192 PRT Thuja plicata 33 Met Ala Ile Trp Asn Gly Arg Val Leu Asn Leu Cys Ile Leu Trp Leu 1 5 10 15 Leu Val Ser Ile Val Leu Leu Asn Gly Ile Asp Cys His Ser Arg Lys 20 25 30 Lys Lys Leu Pro Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Ile Tyr Asn Gly Lys Asn Ala Gly Asn Ala Thr Ser Thr Leu Val 50 55 60 Ala Ala Pro Gln Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Tyr His 65 70 75 80 Phe Gly Asp Leu Ser Val Phe Asp Asp Pro Ile Thr Val Asp Asn Asn 85 90 95 Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Gly Gly Ala Asp Pro Ile 130 135 140 Leu Ala Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Thr Ile Asp Thr Asp Ala Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 34 890 DNA Thuja plicata CDS (44)..(619) 34 cagagctctt ctttcttcaa acatttttga tatattttgc aca atg gca atc tgg 55 Met Ala Ile Trp 1 aat gga aga gtt ctg aat ttg tgc att ctg tgg ctt ctg gtc tcc ata 103 Asn Gly Arg Val Leu Asn Leu Cys Ile Leu Trp Leu Leu Val Ser Ile 5 10 15 20 gtt ttg ctg aat ggt ata gat tgc cat agt aga aaa aag aag ctt cca 151 Val Leu Leu Asn Gly Ile Asp Cys His Ser Arg Lys Lys Lys Leu Pro 25 30 35 aag cca tgt agg aat ctt gtt ttg tat ttt cat gat att atc tac aat 199 Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp Ile Ile Tyr Asn 40 45 50 ggt aaa aat gca ggc aat gca aca tct acg ctt gtt gca gcc cct caa 247 Gly Lys Asn Ala Gly Asn Ala Thr Ser Thr Leu Val Ala Ala Pro Gln 55 60 65 gga gct aat ctc acc att atg act ggc aat tac cat ttt gga gat ctg 295 Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Tyr His Phe Gly Asp Leu 70 75 80 gct gtg ttt gat gat cct att act gtt gac aac aat ctt cat tct cct 343 Ala Val Phe Asp Asp Pro Ile Thr Val Asp Asn Asn Leu His Ser Pro 85 90 95 100 cct gtg gga aga gct cag ggc ttt tac ttc tat gac atg aag aat aca 391 Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Asn Thr 105 110 115 ttc agt gct tgg ctt ggg ttc aca ttt gtg ctg aac tca aca gat tat 439 Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn Ser Thr Asp Tyr 120 125 130 aaa ggc act att act ttc ggt gga gca gac cca att ttg gct aag tac 487 Lys Gly Thr Ile Thr Phe Gly Gly Ala Asp Pro Ile Leu Ala Lys Tyr 135 140 145 aga gat ata tct gtt gtg ggt ggt act gga gat ttc ttg atg gca aga 535 Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe Leu Met Ala Arg 150 155 160 gga att gct aca atc gat act gat gca tat gag gga gat gtt tat ttc 583 Gly Ile Ala Thr Ile Asp Thr Asp Ala Tyr Glu Gly Asp Val Tyr Phe 165 170 175 180 agg cta agg gtg aat atc aca ctc tat gag tgt tac tgatccatgg 629 Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 185 190 gtattctatg tagaatagct caatctgata tggctatatt attttgagag cataggtagt 689 taagttttat aactaagtag tgaaccatga gatcattgaa aacttgggtg ctcatgcaca 749 gttttcatat tttctaaata agtctgctcg actattacat ttatggattg ttgagaattg 809 tgtcgcttat tactttatga ataagctatt ttaaacaaag ttttcacaag tttaaaagtt 869 gtcaaaaaaa aaaaaaaaaa a 890 35 192 PRT Thuja plicata 35 Met Ala Ile Trp Asn Gly Arg Val Leu Asn Leu Cys Ile Leu Trp Leu 1 5 10 15 Leu Val Ser Ile Val Leu Leu Asn Gly Ile Asp Cys His Ser Arg Lys 20 25 30 Lys Lys Leu Pro Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Ile Tyr Asn Gly Lys Asn Ala Gly Asn Ala Thr Ser Thr Leu Val 50 55 60 Ala Ala Pro Gln Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Tyr His 65 70 75 80 Phe Gly Asp Leu Ala Val Phe Asp Asp Pro Ile Thr Val Asp Asn Asn 85 90 95 Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Gly Gly Ala Asp Pro Ile 130 135 140 Leu Ala Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Thr Ile Asp Thr Asp Ala Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 36 30 PRT Forsythia x intermedia 36 Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly Arg 1 5 10 15 Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr 20 25 30 37 16 PRT Forsythia x intermedia PEPTIDE (1)..(16) Peptide fragment, wherein Xaa = unknown amino acid 37 Phe Met Asp Ile Ala Met Xaa Pro Gly Lys Val Thr Leu Asp Glu Lys 1 5 10 15 38 13 PRT Forsythia x intermedia PEPTIDE (1)..(13) Peptide fragment, wherein Xaa = unknown amino acid 38 Leu Pro Xaa Glu Phe Gly Met Asp Pro Ala Lys Phe Met 1 5 10 39 8 PRT Forsythia x intermedia PEPTIDE (1)..(8) Peptide fragment, wherein Xaa = unknown amino acid 39 Glu Val Val Gln Xaa Xaa Glu Lys 1 5 40 10 PRT Forsythia x intermedia PEPTIDE (1)..(10) Peptide fragment, wherein Xaa = unknown amino acid 40 Tyr Xaa Ser Val Glu Glu Tyr Leu Lys Arg 1 5 10 41 12 PRT Forsythia x intermedia 41 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met 1 5 10 42 7 PRT Forsythia x intermedia 42 Met Asp Pro Ala Lys Phe Met 1 5 43 7 PRT Forsythia x intermedia 43 Met Leu Ile Ser Phe Lys Met 1 5 44 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 44 athathggng gnacnggnta 20 45 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 45 gytccatngc natrtccat 19 46 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 46 tcytcnarng tnacyttncc 20 47 1060 DNA Forsythia x intermedia CDS (28)..(963) 47 aattcggcac gagaaaaaca gagagag atg gga aaa agc aaa gtt ttg atc att 54 Met Gly Lys Ser Lys Val Leu Ile Ile 1 5 ggg ggt aca ggg tac tta ggg agg aga ttg gtt aag gca agt tta gct 102 Gly Gly Thr Gly Tyr Leu Gly Arg Arg Leu Val Lys Ala Ser Leu Ala 10 15 20 25 caa ggt cat gaa aca tac att ctg cat agg cct gaa att ggt gtt gat 150 Gln Gly His Glu Thr Tyr Ile Leu His Arg Pro Glu Ile Gly Val Asp 30 35 40 att gat aaa gtt gaa atg cta ata tca ttt aaa atg caa gga gct cat 198 Ile Asp Lys Val Glu Met Leu Ile Ser Phe Lys Met Gln Gly Ala His 45 50 55 ctt gta tct ggt tct ttc aag gat ttc aac agt ctg gtc gag gct gtc 246 Leu Val Ser Gly Ser Phe Lys Asp Phe Asn Ser Leu Val Glu Ala Val 60 65 70 aag ctc gta gac gta gta atc agc gcc att tct ggt gtt cat att cga 294 Lys Leu Val Asp Val Val Ile Ser Ala Ile Ser Gly Val His Ile Arg 75 80 85 agc cat caa att ctt ctt caa ctc aag ctt gtt gaa gct att aaa gag 342 Ser His Gln Ile Leu Leu Gln Leu Lys Leu Val Glu Ala Ile Lys Glu 90 95 100 105 gct gga aat gtc aag aga ttt tta cca tct gag ttt gga atg gat cct 390 Ala Gly Asn Val Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp Pro 110 115 120 gca aaa ttt atg gat acg gcc atg gaa ccc gga aag gta aca ctt gat 438 Ala Lys Phe Met Asp Thr Ala Met Glu Pro Gly Lys Val Thr Leu Asp 125 130 135 gag aag atg gtg gta agg aaa gca att gaa aag gct ggg att cct ttc 486 Glu Lys Met Val Val Arg Lys Ala Ile Glu Lys Ala Gly Ile Pro Phe 140 145 150 aca tat gtc tct gca aat tgc ttt gct ggt tat ttc ttg gga ggt ctc 534 Thr Tyr Val Ser Ala Asn Cys Phe Ala Gly Tyr Phe Leu Gly Gly Leu 155 160 165 tgt caa ttt ggc aaa att ctt cct tct aga gat ttt gtc att ata cat 582 Cys Gln Phe Gly Lys Ile Leu Pro Ser Arg Asp Phe Val Ile Ile His 170 175 180 185 gga gat ggt aac aaa aaa gca ata tat aac aat gaa gat gat ata gca 630 Gly Asp Gly Asn Lys Lys Ala Ile Tyr Asn Asn Glu Asp Asp Ile Ala 190 195 200 act tat gcc atc aaa aca att aat gat cca aga acc ctc aac aag aca 678 Thr Tyr Ala Ile Lys Thr Ile Asn Asp Pro Arg Thr Leu Asn Lys Thr 205 210 215 atc tac att agt cct cca aaa aac atc ctt tca caa aga gaa gtt gtt 726 Ile Tyr Ile Ser Pro Pro Lys Asn Ile Leu Ser Gln Arg Glu Val Val 220 225 230 cag aca tgg gag aag ctt att ggg aaa gaa ctg cag aaa att aca ctc 774 Gln Thr Trp Glu Lys Leu Ile Gly Lys Glu Leu Gln Lys Ile Thr Leu 235 240 245 tcg aag gaa gat ttt tta gcc tcc gtg aaa gag ctc gag tat gct cag 822 Ser Lys Glu Asp Phe Leu Ala Ser Val Lys Glu Leu Glu Tyr Ala Gln 250 255 260 265 caa gtg gga tta agc cat tat cat gat gtc aac tat cag gga tgc ctt 870 Gln Val Gly Leu Ser His Tyr His Asp Val Asn Tyr Gln Gly Cys Leu 270 275 280 acg agt ttt gag ata gga gat gaa gaa gag gca tct aaa ctt tat cca 918 Thr Ser Phe Glu Ile Gly Asp Glu Glu Glu Ala Ser Lys Leu Tyr Pro 285 290 295 gag gtt aag tat acc agt gtg gaa gag tac ctc aag cgt tac gtg 963 Glu Val Lys Tyr Thr Ser Val Glu Glu Tyr Leu Lys Arg Tyr Val 300 305 310 tagttgaaag ctttccatta ttattgtaat aatatttaaa tcagtatgta gttttaaatt 1023 tcgttaaata atatgtgttg aattttgctt ccaaaaa 1060 48 312 PRT Forsythia x intermedia 48 Met Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly 1 5 10 15 Arg Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile 20 25 30 Leu His Arg Pro Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu 35 40 45 Ile Ser Phe Lys Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys 50 55 60 Asp Phe Asn Ser Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Ser Ala Ile Ser Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala 115 120 125 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys 130 135 140 Ala Ile Glu Lys Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys 145 150 155 160 Phe Ala Gly Tyr Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu 165 170 175 Pro Ser Arg Asp Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala 180 185 190 Ile Tyr Asn Asn Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile 195 200 205 Asn Asp Pro Arg Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys 210 215 220 Asn Ile Leu Ser Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile 225 230 235 240 Gly Lys Glu Leu Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala 245 250 255 Ser Val Lys Glu Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr 260 265 270 His Asp Val Asn Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp 275 280 285 Glu Glu Glu Ala Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val 290 295 300 Glu Glu Tyr Leu Lys Arg Tyr Val 305 310 49 1112 DNA Forsythia x intermedia CDS (44)..(979) 49 aattcggcac gagctcgtgc cgcacagaga aaaacagaga gag atg gga aaa agc 55 Met Gly Lys Ser 1 aaa gtt ttg atc att ggg ggt aca ggg tac tta ggg agg aga ttg gtt 103 Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly Arg Arg Leu Val 5 10 15 20 aag gca agt tta gct caa ggt cat gaa aca tac att ctg cat agg cct 151 Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile Leu His Arg Pro 25 30 35 gaa att ggt gtt gat att gat aaa gtt gaa atg cta ata tca ttt aaa 199 Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu Ile Ser Phe Lys 40 45 50 atg caa gga gct cat ctt gta tct ggt tct ttc aag gat ttc aac agt 247 Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys Asp Phe Asn Ser 55 60 65 ctg gtc gag gct gtc aag ctc gta gac gta gta atc agc gcc att tct 295 Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile Ser Ala Ile Ser 70 75 80 ggt gtt cat att cga agc cat caa att ctt ctt caa ctc aag ctt gtt 343 Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln Leu Lys Leu Val 85 90 95 100 gaa gct att aaa gag gct gga aat gtc aag aga ttt tta cca tct gag 391 Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe Leu Pro Ser Glu 105 110 115 ttt gga atg gat cct gca aaa ttt atg gat acg gcc atg gaa ccc gga 439 Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala Met Glu Pro Gly 120 125 130 aag gta aca ctt gat gag aag atg gtg gta agg aaa gca att gaa aag 487 Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys Ala Ile Glu Lys 135 140 145 gct ggg att cct ttc aca tat gtc tct gca aat tgc ttt gct ggt tat 535 Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys Phe Ala Gly Tyr 150 155 160 ttc ttg gga ggt ctc tgt caa ttt ggc aaa att ctt cct tct aga gat 583 Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu Pro Ser Arg Asp 165 170 175 180 ttt gtc att ata cat gga gat ggt aac aaa aaa gca ata tat aac aat 631 Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala Ile Tyr Asn Asn 185 190 195 gaa gat gat ata gca act tat gcc atc aaa aca att aat gat cca aga 679 Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile Asn Asp Pro Arg 200 205 210 acc ctc aac aag aca atc tac att agt cct cca aaa aac atc ctt tca 727 Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys Asn Ile Leu Ser 215 220 225 caa aga gaa gtt gtt cag aca tgg gag aag ctt att ggg aaa gaa ctg 775 Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile Gly Lys Glu Leu 230 235 240 cag aaa att aca ctc tcg aag gaa gat ttt tta gcc tcc gtg aaa gag 823 Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala Ser Val Lys Glu 245 250 255 260 ctc gag tat gct cag caa gtg gga tta agc cat tat cat gat gtc aac 871 Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr His Asp Val Asn 265 270 275 tat cag gga tgc ctt acg agt ttt gag ata gga gat gaa gaa gag gca 919 Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp Glu Glu Glu Ala 280 285 290 tct aaa ctt tat cca gag gtt aag tat acc agt gtg gaa gag tac ctc 967 Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val Glu Glu Tyr Leu 295 300 305 aag cgt tac gtg tagttgaaag ctttccatta ttattgtaat aatatttaaa 1019 Lys Arg Tyr Val 310 tcagtatgta gttttaaatt tcgttaaata atatgtgttg aattttgctt caaacgagtg 1079 gtcgattgaa atggaatttt gaagtcaaaa aaa 1112 50 312 PRT Forsythia x intermedia 50 Met Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly 1 5 10 15 Arg Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile 20 25 30 Leu His Arg Pro Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu 35 40 45 Ile Ser Phe Lys Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys 50 55 60 Asp Phe Asn Ser Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Ser Ala Ile Ser Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala 115 120 125 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys 130 135 140 Ala Ile Glu Lys Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys 145 150 155 160 Phe Ala Gly Tyr Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu 165 170 175 Pro Ser Arg Asp Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala 180 185 190 Ile Tyr Asn Asn Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile 195 200 205 Asn Asp Pro Arg Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys 210 215 220 Asn Ile Leu Ser Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile 225 230 235 240 Gly Lys Glu Leu Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala 245 250 255 Ser Val Lys Glu Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr 260 265 270 His Asp Val Asn Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp 275 280 285 Glu Glu Glu Ala Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val 290 295 300 Glu Glu Tyr Leu Lys Arg Tyr Val 305 310 51 1124 DNA Forsythia x intermedia CDS (29)..(964) 51 aattcggcac gaggaaaaac agagagag atg gga aaa agc aaa gtt ttg atc 52 Met Gly Lys Ser Lys Val Leu Ile 1 5 att ggg ggt aca ggg tac tta ggg agg aga ttg gtt aag gca agt tta 100 Ile Gly Gly Thr Gly Tyr Leu Gly Arg Arg Leu Val Lys Ala Ser Leu 10 15 20 gct caa ggt cat gaa aca tac att ctg cat agg cct gaa att ggt gtt 148 Ala Gln Gly His Glu Thr Tyr Ile Leu His Arg Pro Glu Ile Gly Val 25 30 35 40 gat att gat aaa gtt gaa atg cta ata tca ttt aaa atg caa gga gct 196 Asp Ile Asp Lys Val Glu Met Leu Ile Ser Phe Lys Met Gln Gly Ala 45 50 55 cat ctt gta tct ggt tct ttc aag gat ttc aac agt ctg gtc gag gct 244 His Leu Val Ser Gly Ser Phe Lys Asp Phe Asn Ser Leu Val Glu Ala 60 65 70 gtc aag ctc gta gac gta gta atc agc gcc att tct ggt gtt cat att 292 Val Lys Leu Val Asp Val Val Ile Ser Ala Ile Ser Gly Val His Ile 75 80 85 cga agc cat caa att ctt ctt caa ctc aag ctt gtt gaa gct att aaa 340 Arg Ser His Gln Ile Leu Leu Gln Leu Lys Leu Val Glu Ala Ile Lys 90 95 100 gag gct gga aat gtc aag aga ttt tta cca tct gag ttt gga atg gat 388 Glu Ala Gly Asn Val Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp 105 110 115 120 cct gca aaa ttt atg gat acg gcc atg gaa ccc gga aag gta aca ctt 436 Pro Ala Lys Phe Met Asp Thr Ala Met Glu Pro Gly Lys Val Thr Leu 125 130 135 gat gag aag atg gtg gta agg aaa gca att gaa aag gct ggg att cct 484 Asp Glu Lys Met Val Val Arg Lys Ala Ile Glu Lys Ala Gly Ile Pro 140 145 150 ttc aca tat gtc tct gca aat tgc ttt gct ggt tat ttc ttg gga ggt 532 Phe Thr Tyr Val Ser Ala Asn Cys Phe Ala Gly Tyr Phe Leu Gly Gly 155 160 165 ctc tgt caa ttt ggc aaa att ctt cct tct aga gat ttt gtc att ata 580 Leu Cys Gln Phe Gly Lys Ile Leu Pro Ser Arg Asp Phe Val Ile Ile 170 175 180 cat gga gat ggt aac aaa aaa gca ata tat aac aat gaa gat gat ata 628 His Gly Asp Gly Asn Lys Lys Ala Ile Tyr Asn Asn Glu Asp Asp Ile 185 190 195 200 gca act tat gcc atc aaa aca att aat gat cca aga acc ctc aac aag 676 Ala Thr Tyr Ala Ile Lys Thr Ile Asn Asp Pro Arg Thr Leu Asn Lys 205 210 215 aca atc tac att agt cct cca aaa aac atc ctt tca caa aga gaa gtt 724 Thr Ile Tyr Ile Ser Pro Pro Lys Asn Ile Leu Ser Gln Arg Glu Val 220 225 230 gtt cag aca tgg gag aag ctt att ggg aaa gaa ctg cag aaa att aca 772 Val Gln Thr Trp Glu Lys Leu Ile Gly Lys Glu Leu Gln Lys Ile Thr 235 240 245 ctc tcg aag gaa gat ttt tta gcc tcc gtg aaa gag ctc gag tat gct 820 Leu Ser Lys Glu Asp Phe Leu Ala Ser Val Lys Glu Leu Glu Tyr Ala 250 255 260 cag caa gtg gga tta agc cat tat cat gat gtc aac tat cag gga tgc 868 Gln Gln Val Gly Leu Ser His Tyr His Asp Val Asn Tyr Gln Gly Cys 265 270 275 280 ctt acg agt ttt gag ata gga gat gaa gaa gag gca tct aaa ctt tat 916 Leu Thr Ser Phe Glu Ile Gly Asp Glu Glu Glu Ala Ser Lys Leu Tyr 285 290 295 cca gag gtt aag tat acc agt gtg gaa gag tac ctc aag cgt tac gtg 964 Pro Glu Val Lys Tyr Thr Ser Val Glu Glu Tyr Leu Lys Arg Tyr Val 300 305 310 tagttgaaag ctttccatta ttattgtaat aatatttaaa tcagtatgta gttttaaatt 1024 tcgttaaata atatgtgttg aattttgctt caaacgagtg gtcgattgaa atggaatttt 1084 gaagtcatct tctccacaat attagtccaa ataaaaaaaa 1124 52 312 PRT Forsythia x intermedia 52 Met Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly 1 5 10 15 Arg Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile 20 25 30 Leu His Arg Pro Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu 35 40 45 Ile Ser Phe Lys Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys 50 55 60 Asp Phe Asn Ser Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Ser Ala Ile Ser Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala 115 120 125 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys 130 135 140 Ala Ile Glu Lys Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys 145 150 155 160 Phe Ala Gly Tyr Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu 165 170 175 Pro Ser Arg Asp Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala 180 185 190 Ile Tyr Asn Asn Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile 195 200 205 Asn Asp Pro Arg Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys 210 215 220 Asn Ile Leu Ser Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile 225 230 235 240 Gly Lys Glu Leu Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala 245 250 255 Ser Val Lys Glu Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr 260 265 270 His Asp Val Asn Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp 275 280 285 Glu Glu Glu Ala Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val 290 295 300 Glu Glu Tyr Leu Lys Arg Tyr Val 305 310 53 1097 DNA Forsythia x intermedia CDS (29)..(964) 53 aattcggcac gaggaaaaac agagagag atg gga aaa agc aaa gtt ttg atc 52 Met Gly Lys Ser Lys Val Leu Ile 1 5 att ggg ggt aca ggg tac tta ggg agg aga ttg gtt aag gca agt tta 100 Ile Gly Gly Thr Gly Tyr Leu Gly Arg Arg Leu Val Lys Ala Ser Leu 10 15 20 gct caa ggt cat gaa aca tac att ctg cat agg cct gaa att ggt gtt 148 Ala Gln Gly His Glu Thr Tyr Ile Leu His Arg Pro Glu Ile Gly Val 25 30 35 40 gat att gat aaa gtt gaa atg cta ata tca ttt aaa atg caa gga gct 196 Asp Ile Asp Lys Val Glu Met Leu Ile Ser Phe Lys Met Gln Gly Ala 45 50 55 cat ctt gta tct ggt tct ttc aag gat ttc aac agt ctg gtc gag gct 244 His Leu Val Ser Gly Ser Phe Lys Asp Phe Asn Ser Leu Val Glu Ala 60 65 70 gtc aag ctc gta gac gta gta atc agc gcc att tct ggt gtt cat att 292 Val Lys Leu Val Asp Val Val Ile Ser Ala Ile Ser Gly Val His Ile 75 80 85 cga agc cat caa att ctt ctt caa ctc aag ctt gtt gaa gct att aaa 340 Arg Ser His Gln Ile Leu Leu Gln Leu Lys Leu Val Glu Ala Ile Lys 90 95 100 gag gct gga aat gtc aag aga ttt tta cca tct gag ttt gga atg gat 388 Glu Ala Gly Asn Val Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp 105 110 115 120 cct gca aaa ttt atg gat acg gcc atg gaa ccc gga aag gta aca ctt 436 Pro Ala Lys Phe Met Asp Thr Ala Met Glu Pro Gly Lys Val Thr Leu 125 130 135 gat gag aag atg gtg gta agg aaa gca att gaa aag gct ggg att cct 484 Asp Glu Lys Met Val Val Arg Lys Ala Ile Glu Lys Ala Gly Ile Pro 140 145 150 ttc aca tat gtc tct gca aat tgc ttt gct ggt tat ttc ttg gga ggt 532 Phe Thr Tyr Val Ser Ala Asn Cys Phe Ala Gly Tyr Phe Leu Gly Gly 155 160 165 ctc tgt caa ttt ggc aaa att ctt cct tct aga gat ttt gtc att ata 580 Leu Cys Gln Phe Gly Lys Ile Leu Pro Ser Arg Asp Phe Val Ile Ile 170 175 180 cat gga gat ggt aac aaa aaa gca ata tat aac aat gaa gat gat ata 628 His Gly Asp Gly Asn Lys Lys Ala Ile Tyr Asn Asn Glu Asp Asp Ile 185 190 195 200 gca act tat gcc atc aaa aca att aat gat cca aga acc ctc aac aag 676 Ala Thr Tyr Ala Ile Lys Thr Ile Asn Asp Pro Arg Thr Leu Asn Lys 205 210 215 aca atc tac att agt cct cca aaa aac atc ctt tca caa aga gaa gtt 724 Thr Ile Tyr Ile Ser Pro Pro Lys Asn Ile Leu Ser Gln Arg Glu Val 220 225 230 gtt cag aca tgg gag aag ctt att ggg aaa gaa ctg cag aaa att aca 772 Val Gln Thr Trp Glu Lys Leu Ile Gly Lys Glu Leu Gln Lys Ile Thr 235 240 245 ctc tcg aag gaa gat ttt tta gcc tcc gtg aaa gag ctc gag tat gct 820 Leu Ser Lys Glu Asp Phe Leu Ala Ser Val Lys Glu Leu Glu Tyr Ala 250 255 260 cag caa gtg gga tta agc cat tat cat gat gtc aac tat cag gga tgc 868 Gln Gln Val Gly Leu Ser His Tyr His Asp Val Asn Tyr Gln Gly Cys 265 270 275 280 ctt acg agt ttt gag ata gga gat gaa gaa gag gca tct aaa ctt tat 916 Leu Thr Ser Phe Glu Ile Gly Asp Glu Glu Glu Ala Ser Lys Leu Tyr 285 290 295 cca gag gtt aag tat acc agt gtg gaa gag tac ctc aag cgt tac gtg 964 Pro Glu Val Lys Tyr Thr Ser Val Glu Glu Tyr Leu Lys Arg Tyr Val 300 305 310 tagttgaaag ctttccatta ttattgtaat aatatttaaa tcagtatgta gttttaaatt 1024 tcgttaaata atatgtgttg aattttgctt caaacgagtg gtcgattgaa atggaatttt 1084 gaaaaaaaaa aaa 1097 54 312 PRT Forsythia x intermedia 54 Met Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly 1 5 10 15 Arg Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile 20 25 30 Leu His Arg Pro Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu 35 40 45 Ile Ser Phe Lys Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys 50 55 60 Asp Phe Asn Ser Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Ser Ala Ile Ser Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala 115 120 125 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys 130 135 140 Ala Ile Glu Lys Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys 145 150 155 160 Phe Ala Gly Tyr Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu 165 170 175 Pro Ser Arg Asp Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala 180 185 190 Ile Tyr Asn Asn Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile 195 200 205 Asn Asp Pro Arg Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys 210 215 220 Asn Ile Leu Ser Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile 225 230 235 240 Gly Lys Glu Leu Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala 245 250 255 Ser Val Lys Glu Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr 260 265 270 His Asp Val Asn Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp 275 280 285 Glu Glu Glu Ala Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val 290 295 300 Glu Glu Tyr Leu Lys Arg Tyr Val 305 310 55 1109 DNA Forsythia x intermedia CDS (31)..(966) 55 aattcggcac gaggagaaaa acagagagag atg gga aaa agc aaa gtt ttg atc 54 Met Gly Lys Ser Lys Val Leu Ile 1 5 att ggg ggt aca ggg tac tta ggg agg aga ttg gtt aag gca agt tta 102 Ile Gly Gly Thr Gly Tyr Leu Gly Arg Arg Leu Val Lys Ala Ser Leu 10 15 20 gct caa ggt cat gaa aca tac att ctg cat agg cct gaa att ggt gtt 150 Ala Gln Gly His Glu Thr Tyr Ile Leu His Arg Pro Glu Ile Gly Val 25 30 35 40 gat att gat aaa gtt gaa atg cta ata tca ttt aaa atg caa gga gct 198 Asp Ile Asp Lys Val Glu Met Leu Ile Ser Phe Lys Met Gln Gly Ala 45 50 55 cat ctt gta tct ggt tct ttc aag gat ttc aac agt ctg gtc gag gct 246 His Leu Val Ser Gly Ser Phe Lys Asp Phe Asn Ser Leu Val Glu Ala 60 65 70 gtc aag ctc gta gac gta gta atc agc gcc att tct ggt gtt cat att 294 Val Lys Leu Val Asp Val Val Ile Ser Ala Ile Ser Gly Val His Ile 75 80 85 cga agc cat caa att ctt ctt caa ctc aag ctt gtt gaa gct att aaa 342 Arg Ser His Gln Ile Leu Leu Gln Leu Lys Leu Val Glu Ala Ile Lys 90 95 100 gag gct gga aat gtc aag aga ttt tta cca tct gag ttt gga atg gat 390 Glu Ala Gly Asn Val Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp 105 110 115 120 cct gca aaa ttt atg gat acg gcc atg gaa ccc gga aag gta aca ctt 438 Pro Ala Lys Phe Met Asp Thr Ala Met Glu Pro Gly Lys Val Thr Leu 125 130 135 gat gag aag atg gtg gta agg aaa gca att gaa aag gct ggg att cct 486 Asp Glu Lys Met Val Val Arg Lys Ala Ile Glu Lys Ala Gly Ile Pro 140 145 150 ttc aca tat gtc tct gca aat tgc ttt gct ggt tat ttc ttg gga ggt 534 Phe Thr Tyr Val Ser Ala Asn Cys Phe Ala Gly Tyr Phe Leu Gly Gly 155 160 165 ctc tgt caa ttt ggc aaa att ctt cct tct aga gat ttt gtc att ata 582 Leu Cys Gln Phe Gly Lys Ile Leu Pro Ser Arg Asp Phe Val Ile Ile 170 175 180 cat gga gat ggt aac aaa aaa gca ata tat aac aat gaa gat gat ata 630 His Gly Asp Gly Asn Lys Lys Ala Ile Tyr Asn Asn Glu Asp Asp Ile 185 190 195 200 gca act tat gcc atc aaa aca att aat gat cca aga acc ctc aac aag 678 Ala Thr Tyr Ala Ile Lys Thr Ile Asn Asp Pro Arg Thr Leu Asn Lys 205 210 215 aca atc tac att agt cct cca aaa aac atc ctt tca caa aga gaa gtt 726 Thr Ile Tyr Ile Ser Pro Pro Lys Asn Ile Leu Ser Gln Arg Glu Val 220 225 230 gtt cag aca tgg gag aag ctt att ggg aaa gaa ctg cag aaa att aca 774 Val Gln Thr Trp Glu Lys Leu Ile Gly Lys Glu Leu Gln Lys Ile Thr 235 240 245 ctc tcg aag gaa gat ttt tta gcc tcc gtg aaa gag ctc gag tat gct 822 Leu Ser Lys Glu Asp Phe Leu Ala Ser Val Lys Glu Leu Glu Tyr Ala 250 255 260 cag caa gtg gga tta agc cat tat cat gat gtc aac tat cag gga tgc 870 Gln Gln Val Gly Leu Ser His Tyr His Asp Val Asn Tyr Gln Gly Cys 265 270 275 280 ctt acg agt ttt gag ata gga gat gaa gaa gag gca tct aaa ctt tat 918 Leu Thr Ser Phe Glu Ile Gly Asp Glu Glu Glu Ala Ser Lys Leu Tyr 285 290 295 cca gag gtt aag tat acc agt gtg gaa gag tac ctc aag cgt tac gtg 966 Pro Glu Val Lys Tyr Thr Ser Val Glu Glu Tyr Leu Lys Arg Tyr Val 300 305 310 tagttgaaag ctttccatta ttattgtaat aatatttaaa tcagtatgta gttttaaatt 1026 tcgttaaata atatgtgttg aattttgctt caaacgagtg gtcgattgaa atggaatttt 1086 gaagtcatct tctccaaaaa aaa 1109 56 312 PRT Forsythia x intermedia 56 Met Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly 1 5 10 15 Arg Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile 20 25 30 Leu His Arg Pro Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu 35 40 45 Ile Ser Phe Lys Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys 50 55 60 Asp Phe Asn Ser Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Ser Ala Ile Ser Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala 115 120 125 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys 130 135 140 Ala Ile Glu Lys Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys 145 150 155 160 Phe Ala Gly Tyr Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu 165 170 175 Pro Ser Arg Asp Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala 180 185 190 Ile Tyr Asn Asn Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile 195 200 205 Asn Asp Pro Arg Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys 210 215 220 Asn Ile Leu Ser Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile 225 230 235 240 Gly Lys Glu Leu Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala 245 250 255 Ser Val Lys Glu Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr 260 265 270 His Asp Val Asn Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp 275 280 285 Glu Glu Glu Ala Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val 290 295 300 Glu Glu Tyr Leu Lys Arg Tyr Val 305 310 57 1107 DNA Forsythia x intermedia CDS (27)..(962) 57 aattcggcac gagaaaacag agagag atg gga aaa agc aaa gtt ttg atc att 53 Met Gly Lys Ser Lys Val Leu Ile Ile 1 5 ggg ggt aca ggg tac tta ggg agg aga ttg gtt aag gca agt tta gct 101 Gly Gly Thr Gly Tyr Leu Gly Arg Arg Leu Val Lys Ala Ser Leu Ala 10 15 20 25 caa ggt cat gaa aca tac att ctg cat agg cct gaa att ggt gtt gat 149 Gln Gly His Glu Thr Tyr Ile Leu His Arg Pro Glu Ile Gly Val Asp 30 35 40 att gat aaa gtt gaa atg cta ata tca ttt aaa atg caa gga gct cat 197 Ile Asp Lys Val Glu Met Leu Ile Ser Phe Lys Met Gln Gly Ala His 45 50 55 ctt gta tct ggt tct ttc aag gat ttc aac agt ctg gtc gag gct gtc 245 Leu Val Ser Gly Ser Phe Lys Asp Phe Asn Ser Leu Val Glu Ala Val 60 65 70 aag ctc gta gac gta gta atc agc gcc att tct ggt gtt cat att cga 293 Lys Leu Val Asp Val Val Ile Ser Ala Ile Ser Gly Val His Ile Arg 75 80 85 agc cat caa att ctt ctt caa ctc aag ctt gtt gaa gct att aaa gag 341 Ser His Gln Ile Leu Leu Gln Leu Lys Leu Val Glu Ala Ile Lys Glu 90 95 100 105 gct gga aat gtc aag aga ttt tta cca tct gag ttt gga atg gat cct 389 Ala Gly Asn Val Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp Pro 110 115 120 gca aaa ttt atg gat acg gcc atg gaa ccc gga aag gta aca ctt gat 437 Ala Lys Phe Met Asp Thr Ala Met Glu Pro Gly Lys Val Thr Leu Asp 125 130 135 gag aag atg gtg gta agg aaa gca att gaa aag gct ggg att cct ttc 485 Glu Lys Met Val Val Arg Lys Ala Ile Glu Lys Ala Gly Ile Pro Phe 140 145 150 aca tat gtc tct gca aat tgc ttt gct ggt tat ttc ttg gga ggt ctc 533 Thr Tyr Val Ser Ala Asn Cys Phe Ala Gly Tyr Phe Leu Gly Gly Leu 155 160 165 tgt caa ttt ggc aaa att ctt cct tct aga gat ttt gtc att ata cat 581 Cys Gln Phe Gly Lys Ile Leu Pro Ser Arg Asp Phe Val Ile Ile His 170 175 180 185 gga gat ggt aac aaa aaa gca ata tat aac aat gaa gat gat ata gca 629 Gly Asp Gly Asn Lys Lys Ala Ile Tyr Asn Asn Glu Asp Asp Ile Ala 190 195 200 act tat gcc atc aaa aca att aat gat cca aga acc ctc aac aag aca 677 Thr Tyr Ala Ile Lys Thr Ile Asn Asp Pro Arg Thr Leu Asn Lys Thr 205 210 215 atc tac att agt cct cca aaa aac atc ctt tca caa aga gaa gtt gtt 725 Ile Tyr Ile Ser Pro Pro Lys Asn Ile Leu Ser Gln Arg Glu Val Val 220 225 230 cag aca tgg gag aag ctt att ggg aaa gaa ctg cag aaa att aca ctc 773 Gln Thr Trp Glu Lys Leu Ile Gly Lys Glu Leu Gln Lys Ile Thr Leu 235 240 245 tcg aag gaa gat ttt tta gcc tcc gtg aaa gag ctc gag tat gct cag 821 Ser Lys Glu Asp Phe Leu Ala Ser Val Lys Glu Leu Glu Tyr Ala Gln 250 255 260 265 caa gtg gga tta agc cat tat cat gat gtc aac tat cag gga tgc ctt 869 Gln Val Gly Leu Ser His Tyr His Asp Val Asn Tyr Gln Gly Cys Leu 270 275 280 acg agt ttt gag ata gga gat gaa gaa gag gca tct aaa ctt tat cca 917 Thr Ser Phe Glu Ile Gly Asp Glu Glu Glu Ala Ser Lys Leu Tyr Pro 285 290 295 gag gtt aag tat acc agt gtg gaa gag tac ctc aag cgt tac gtg 962 Glu Val Lys Tyr Thr Ser Val Glu Glu Tyr Leu Lys Arg Tyr Val 300 305 310 tagttgaaag ctttccatta ttattgtaat aatatttaaa tcagtatgta gttttaaatt 1022 tcgttaaata atatgtgttg aattttgctt caaacgagtg gtcgattgaa atggaatttt 1082 gaagtcatct tctccacaaa aaaaa 1107 58 312 PRT Forsythia x intermedia 58 Met Gly Lys Ser Lys Val Leu Ile Ile Gly Gly Thr Gly Tyr Leu Gly 1 5 10 15 Arg Arg Leu Val Lys Ala Ser Leu Ala Gln Gly His Glu Thr Tyr Ile 20 25 30 Leu His Arg Pro Glu Ile Gly Val Asp Ile Asp Lys Val Glu Met Leu 35 40 45 Ile Ser Phe Lys Met Gln Gly Ala His Leu Val Ser Gly Ser Phe Lys 50 55 60 Asp Phe Asn Ser Leu Val Glu Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Ser Ala Ile Ser Gly Val His Ile Arg Ser His Gln Ile Leu Leu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Ala Lys Phe Met Asp Thr Ala 115 120 125 Met Glu Pro Gly Lys Val Thr Leu Asp Glu Lys Met Val Val Arg Lys 130 135 140 Ala Ile Glu Lys Ala Gly Ile Pro Phe Thr Tyr Val Ser Ala Asn Cys 145 150 155 160 Phe Ala Gly Tyr Phe Leu Gly Gly Leu Cys Gln Phe Gly Lys Ile Leu 165 170 175 Pro Ser Arg Asp Phe Val Ile Ile His Gly Asp Gly Asn Lys Lys Ala 180 185 190 Ile Tyr Asn Asn Glu Asp Asp Ile Ala Thr Tyr Ala Ile Lys Thr Ile 195 200 205 Asn Asp Pro Arg Thr Leu Asn Lys Thr Ile Tyr Ile Ser Pro Pro Lys 210 215 220 Asn Ile Leu Ser Gln Arg Glu Val Val Gln Thr Trp Glu Lys Leu Ile 225 230 235 240 Gly Lys Glu Leu Gln Lys Ile Thr Leu Ser Lys Glu Asp Phe Leu Ala 245 250 255 Ser Val Lys Glu Leu Glu Tyr Ala Gln Gln Val Gly Leu Ser His Tyr 260 265 270 His Asp Val Asn Tyr Gln Gly Cys Leu Thr Ser Phe Glu Ile Gly Asp 275 280 285 Glu Glu Glu Ala Ser Lys Leu Tyr Pro Glu Val Lys Tyr Thr Ser Val 290 295 300 Glu Glu Tyr Leu Lys Arg Tyr Val 305 310 59 26 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 59 gtctcgagtt tttttttttt tttttt 26 60 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 60 gcacataaga gtatggataa g 21 61 1190 DNA Thuja plicata CDS (13)..(951) 61 gcacataaga gt atg gat aag aag agc aga gtt ctg ata gtg ggg ggc act 51 Met Asp Lys Lys Ser Arg Val Leu Ile Val Gly Gly Thr 1 5 10 ggt tat ata ggc aaa aga att gtg aat gcc agt ata tct ctt ggc cat 99 Gly Tyr Ile Gly Lys Arg Ile Val Asn Ala Ser Ile Ser Leu Gly His 15 20 25 ccc act tat gtt ttg ttc aga cca gaa gtg gtc tct aac att gac aaa 147 Pro Thr Tyr Val Leu Phe Arg Pro Glu Val Val Ser Asn Ile Asp Lys 30 35 40 45 gtg cag atg ctg tta tac ttc aaa cag ctt ggt gcc aaa ctt att gag 195 Val Gln Met Leu Leu Tyr Phe Lys Gln Leu Gly Ala Lys Leu Ile Glu 50 55 60 gct tca ttg gat gac cac caa agg ctt gtg gat gct ctg aaa caa gtg 243 Ala Ser Leu Asp Asp His Gln Arg Leu Val Asp Ala Leu Lys Gln Val 65 70 75 gat gtt gtc ata agt gct ttg gca gga ggt gtt cta agc cac cat ata 291 Asp Val Val Ile Ser Ala Leu Ala Gly Gly Val Leu Ser His His Ile 80 85 90 ctt gaa cag ctc aaa cta gtg gaa gcc atc aaa gaa gct gga aat att 339 Leu Glu Gln Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile 95 100 105 aag aga ttt ctt cca tct gag ttt ggc atg gat cca gat att atg gag 387 Lys Arg Phe Leu Pro Ser Glu Phe Gly Met Asp Pro Asp Ile Met Glu 110 115 120 125 cat gca ttg caa cct ggt agc att aca ttc atc gat aag aga aag gtt 435 His Ala Leu Gln Pro Gly Ser Ile Thr Phe Ile Asp Lys Arg Lys Val 130 135 140 cgg cgt gcc att gaa gca gca tcc att cct tac aca tat gtg tct tca 483 Arg Arg Ala Ile Glu Ala Ala Ser Ile Pro Tyr Thr Tyr Val Ser Ser 145 150 155 aat atg ttt gct ggt tac ttt gct gga agt tta gct caa ctt gat ggt 531 Asn Met Phe Ala Gly Tyr Phe Ala Gly Ser Leu Ala Gln Leu Asp Gly 160 165 170 cat atg atg cct cct cga gac aag gtc ctc atc tat gga gat gga aat 579 His Met Met Pro Pro Arg Asp Lys Val Leu Ile Tyr Gly Asp Gly Asn 175 180 185 gtt aaa ggt att tgg gtg gat gaa gat gat gtt gga aca tac aca atc 627 Val Lys Gly Ile Trp Val Asp Glu Asp Asp Val Gly Thr Tyr Thr Ile 190 195 200 205 aaa tca att gat gat cca caa acc ctt aac aag act atg tat att agg 675 Lys Ser Ile Asp Asp Pro Gln Thr Leu Asn Lys Thr Met Tyr Ile Arg 210 215 220 cca cct atg aat atc ctt tca cag aag gaa gtt ata caa ata tgg gag 723 Pro Pro Met Asn Ile Leu Ser Gln Lys Glu Val Ile Gln Ile Trp Glu 225 230 235 aga tta tca gaa caa aac ctg gat aaa ata tac att tct tct caa gac 771 Arg Leu Ser Glu Gln Asn Leu Asp Lys Ile Tyr Ile Ser Ser Gln Asp 240 245 250 ttt ctt gca gat atg aaa gat aaa tca tat gaa gag aag att gta cga 819 Phe Leu Ala Asp Met Lys Asp Lys Ser Tyr Glu Glu Lys Ile Val Arg 255 260 265 tgt cat ctc tac caa att ttc ttt aga gga gat ctt tac aac ttt gaa 867 Cys His Leu Tyr Gln Ile Phe Phe Arg Gly Asp Leu Tyr Asn Phe Glu 270 275 280 285 att ggc ccc aat gct att gaa gct acc aaa ctt tat cca gaa gtg aaa 915 Ile Gly Pro Asn Ala Ile Glu Ala Thr Lys Leu Tyr Pro Glu Val Lys 290 295 300 tac gta acc atg gat tca tat tta gag cgc tat gtt tgaatatctt 961 Tyr Val Thr Met Asp Ser Tyr Leu Glu Arg Tyr Val 305 310 tctagttttg tatattgttt ttctacatga taatgtgaga ggtactattt caaataattt 1021 agacttatgg ctcaatttta aaactagagt acactttatt ccaaattact tacactattt 1081 tttacttcat attgtactca atatagactt ggtataaaga atatggaatc ataatgatat 1141 tataattatt tatagatctt attttaaata aaaaaaaaaa aaaaaaaaa 1190 62 313 PRT Thuja plicata 62 Met Asp Lys Lys Ser Arg Val Leu Ile Val Gly Gly Thr Gly Tyr Ile 1 5 10 15 Gly Lys Arg Ile Val Asn Ala Ser Ile Ser Leu Gly His Pro Thr Tyr 20 25 30 Val Leu Phe Arg Pro Glu Val Val Ser Asn Ile Asp Lys Val Gln Met 35 40 45 Leu Leu Tyr Phe Lys Gln Leu Gly Ala Lys Leu Ile Glu Ala Ser Leu 50 55 60 Asp Asp His Gln Arg Leu Val Asp Ala Leu Lys Gln Val Asp Val Val 65 70 75 80 Ile Ser Ala Leu Ala Gly Gly Val Leu Ser His His Ile Leu Glu Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Asp Ile Met Glu His Ala Leu 115 120 125 Gln Pro Gly Ser Ile Thr Phe Ile Asp Lys Arg Lys Val Arg Arg Ala 130 135 140 Ile Glu Ala Ala Ser Ile Pro Tyr Thr Tyr Val Ser Ser Asn Met Phe 145 150 155 160 Ala Gly Tyr Phe Ala Gly Ser Leu Ala Gln Leu Asp Gly His Met Met 165 170 175 Pro Pro Arg Asp Lys Val Leu Ile Tyr Gly Asp Gly Asn Val Lys Gly 180 185 190 Ile Trp Val Asp Glu Asp Asp Val Gly Thr Tyr Thr Ile Lys Ser Ile 195 200 205 Asp Asp Pro Gln Thr Leu Asn Lys Thr Met Tyr Ile Arg Pro Pro Met 210 215 220 Asn Ile Leu Ser Gln Lys Glu Val Ile Gln Ile Trp Glu Arg Leu Ser 225 230 235 240 Glu Gln Asn Leu Asp Lys Ile Tyr Ile Ser Ser Gln Asp Phe Leu Ala 245 250 255 Asp Met Lys Asp Lys Ser Tyr Glu Glu Lys Ile Val Arg Cys His Leu 260 265 270 Tyr Gln Ile Phe Phe Arg Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro 275 280 285 Asn Ala Ile Glu Ala Thr Lys Leu Tyr Pro Glu Val Lys Tyr Val Thr 290 295 300 Met Asp Ser Tyr Leu Glu Arg Tyr Val 305 310 63 1151 DNA Thuja plicata CDS (61)..(996) 63 gataagcagc atttcttcac caaagtggtc cgccattaaa ggaatagttt gaaagcagag 60 atg gaa gag agt agc agg gtt ttg ata gtg gga ggc aca gga tac ata 108 Met Glu Glu Ser Ser Arg Val Leu Ile Val Gly Gly Thr Gly Tyr Ile 1 5 10 15 ggc aga agg att gtg aaa gcc agc att gct ctg ggc cat cct act ttc 156 Gly Arg Arg Ile Val Lys Ala Ser Ile Ala Leu Gly His Pro Thr Phe 20 25 30 att ttg ttt agg aaa gaa gtt gtt tct gat gta gag aaa gtg gag atg 204 Ile Leu Phe Arg Lys Glu Val Val Ser Asp Val Glu Lys Val Glu Met 35 40 45 tta ttg tcc ttc aaa aag aat ggt gcc aaa tta ctg gag gct tca ttt 252 Leu Leu Ser Phe Lys Lys Asn Gly Ala Lys Leu Leu Glu Ala Ser Phe 50 55 60 gat gat cac gaa agc ctt gta gat gct gtg aag cag gtt gat gtt gtg 300 Asp Asp His Glu Ser Leu Val Asp Ala Val Lys Gln Val Asp Val Val 65 70 75 80 ata agt gca gtt gca gga aac cac atg cgg cat cac atc ctt caa cag 348 Ile Ser Ala Val Ala Gly Asn His Met Arg His His Ile Leu Gln Gln 85 90 95 ctc aaa tta gtg gag gcc att aaa gaa gct gga aat att aag agg ttt 396 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe 100 105 110 gtt cct tca gaa ttt ggg atg gat cca ggg tta atg gag cat gca atg 444 Val Pro Ser Glu Phe Gly Met Asp Pro Gly Leu Met Glu His Ala Met 115 120 125 gca cct ggc aac att gta ttt att gat aaa ata aaa gtt cga gag gcc 492 Ala Pro Gly Asn Ile Val Phe Ile Asp Lys Ile Lys Val Arg Glu Ala 130 135 140 ata gaa gct gca tcc att cct cac act tat atc tct gcc aac ata ttt 540 Ile Glu Ala Ala Ser Ile Pro His Thr Tyr Ile Ser Ala Asn Ile Phe 145 150 155 160 gct ggc tac ttg gtt ggt gga tta gct caa ctt ggt cgt gtg atg cct 588 Ala Gly Tyr Leu Val Gly Gly Leu Ala Gln Leu Gly Arg Val Met Pro 165 170 175 cct tca gaa aaa gta att ctc tat gga gat gga aat gtc aaa gct gtt 636 Pro Ser Glu Lys Val Ile Leu Tyr Gly Asp Gly Asn Val Lys Ala Val 180 185 190 tgg gta gat gaa gat gat gtt gga ata tac aca atc aaa gca att gat 684 Trp Val Asp Glu Asp Asp Val Gly Ile Tyr Thr Ile Lys Ala Ile Asp 195 200 205 gac cct cac acc cta aat aag act atg tac atc agg cca cct ttg aat 732 Asp Pro His Thr Leu Asn Lys Thr Met Tyr Ile Arg Pro Pro Leu Asn 210 215 220 att ctt tct cag aag gaa gtg gtt gaa aaa tgg gaa aag tta tca gga 780 Ile Leu Ser Gln Lys Glu Val Val Glu Lys Trp Glu Lys Leu Ser Gly 225 230 235 240 aag agc tta aat aaa ata aat att tct gtt gaa gat ttt ctt gca ggc 828 Lys Ser Leu Asn Lys Ile Asn Ile Ser Val Glu Asp Phe Leu Ala Gly 245 250 255 atg gaa ggt caa tca tat gga gag cag att gga ata tca cat ttc tac 876 Met Glu Gly Gln Ser Tyr Gly Glu Gln Ile Gly Ile Ser His Phe Tyr 260 265 270 caa atg ttc tat agg ggt gat ctt tat aat ttt gaa att gga cct aat 924 Gln Met Phe Tyr Arg Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asn 275 280 285 gga gta gaa gct tcc caa ctt tat cca gaa gta aaa tat aca aca gtg 972 Gly Val Glu Ala Ser Gln Leu Tyr Pro Glu Val Lys Tyr Thr Thr Val 290 295 300 gat tca tac atg gaa cgc tac cta tgaaaatctt cttcacgaag atatctaaat 1026 Asp Ser Tyr Met Glu Arg Tyr Leu 305 310 ttaatttaag ctttctaaaa gtttttatat tttgacatta tgctaaataa aaatggagag 1086 tatctagata ataatattga ccaatcatat taaaaattat tgggattaaa aaaaaaaaaa 1146 aaaaa 1151 64 312 PRT Thuja plicata 64 Met Glu Glu Ser Ser Arg Val Leu Ile Val Gly Gly Thr Gly Tyr Ile 1 5 10 15 Gly Arg Arg Ile Val Lys Ala Ser Ile Ala Leu Gly His Pro Thr Phe 20 25 30 Ile Leu Phe Arg Lys Glu Val Val Ser Asp Val Glu Lys Val Glu Met 35 40 45 Leu Leu Ser Phe Lys Lys Asn Gly Ala Lys Leu Leu Glu Ala Ser Phe 50 55 60 Asp Asp His Glu Ser Leu Val Asp Ala Val Lys Gln Val Asp Val Val 65 70 75 80 Ile Ser Ala Val Ala Gly Asn His Met Arg His His Ile Leu Gln Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe 100 105 110 Val Pro Ser Glu Phe Gly Met Asp Pro Gly Leu Met Glu His Ala Met 115 120 125 Ala Pro Gly Asn Ile Val Phe Ile Asp Lys Ile Lys Val Arg Glu Ala 130 135 140 Ile Glu Ala Ala Ser Ile Pro His Thr Tyr Ile Ser Ala Asn Ile Phe 145 150 155 160 Ala Gly Tyr Leu Val Gly Gly Leu Ala Gln Leu Gly Arg Val Met Pro 165 170 175 Pro Ser Glu Lys Val Ile Leu Tyr Gly Asp Gly Asn Val Lys Ala Val 180 185 190 Trp Val Asp Glu Asp Asp Val Gly Ile Tyr Thr Ile Lys Ala Ile Asp 195 200 205 Asp Pro His Thr Leu Asn Lys Thr Met Tyr Ile Arg Pro Pro Leu Asn 210 215 220 Ile Leu Ser Gln Lys Glu Val Val Glu Lys Trp Glu Lys Leu Ser Gly 225 230 235 240 Lys Ser Leu Asn Lys Ile Asn Ile Ser Val Glu Asp Phe Leu Ala Gly 245 250 255 Met Glu Gly Gln Ser Tyr Gly Glu Gln Ile Gly Ile Ser His Phe Tyr 260 265 270 Gln Met Phe Tyr Arg Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asn 275 280 285 Gly Val Glu Ala Ser Gln Leu Tyr Pro Glu Val Lys Tyr Thr Thr Val 290 295 300 Asp Ser Tyr Met Glu Arg Tyr Leu 305 310 65 1308 DNA Thuja plicata CDS (164)..(1105) 65 aaaaactctt agacttattt tcatttttac ccagttcata agtgtttgtt gggtctcttc 60 aaaaaaagcc ccctctcgtt agaggcaaag aacagcatgc tcagatatat gtaagaagca 120 aaatgcccaa aatttgactg tgaaagtgga tgcacataag aat atg gat aag aag 175 Met Asp Lys Lys 1 agc aga gtt cta ata gtg ggg ggt act ggt ttt ata ggc aaa aga att 223 Ser Arg Val Leu Ile Val Gly Gly Thr Gly Phe Ile Gly Lys Arg Ile 5 10 15 20 gtg aag gcc agt ttg gct ctt ggc cat cct act tat gtt ttg ttc agg 271 Val Lys Ala Ser Leu Ala Leu Gly His Pro Thr Tyr Val Leu Phe Arg 25 30 35 cca gaa gcc ctc tct tac att gac aaa gtg cag atg ttg ata tcc ttc 319 Pro Glu Ala Leu Ser Tyr Ile Asp Lys Val Gln Met Leu Ile Ser Phe 40 45 50 aaa cag ctt ggg gcc aaa ctt ctt gag gct tca ttg gat gac cac caa 367 Lys Gln Leu Gly Ala Lys Leu Leu Glu Ala Ser Leu Asp Asp His Gln 55 60 65 ggg ctt gtg gat gtt gtg aaa caa gta gat gtt gtg atc agt gct gtt 415 Gly Leu Val Asp Val Val Lys Gln Val Asp Val Val Ile Ser Ala Val 70 75 80 tca gga ggt ctg gtg cgc cac cat ata ctt gac cag ctc aag cta gtg 463 Ser Gly Gly Leu Val Arg His His Ile Leu Asp Gln Leu Lys Leu Val 85 90 95 100 gag gca att aaa gaa gct ggc aat att aag aga ttt ctt cct tca gaa 511 Glu Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe Leu Pro Ser Glu 105 110 115 ttt ggg atg gac cca gat gtt gta gaa gat cca ttg gaa cct ggt aac 559 Phe Gly Met Asp Pro Asp Val Val Glu Asp Pro Leu Glu Pro Gly Asn 120 125 130 att aca ttc att gat aaa aga aaa gtt aga cgt gcc att gaa gca gca 607 Ile Thr Phe Ile Asp Lys Arg Lys Val Arg Arg Ala Ile Glu Ala Ala 135 140 145 acc att cct tac aca tat gtg tct tca aat atg ttt gct ggg ttc ttt 655 Thr Ile Pro Tyr Thr Tyr Val Ser Ser Asn Met Phe Ala Gly Phe Phe 150 155 160 gct gga agc tta gca caa ctg caa gat gct ccc cgc atg atg cct gct 703 Ala Gly Ser Leu Ala Gln Leu Gln Asp Ala Pro Arg Met Met Pro Ala 165 170 175 180 cga gat aaa gtt ctc ata tat gga gat gga aat gtt aaa ggt gtt tat 751 Arg Asp Lys Val Leu Ile Tyr Gly Asp Gly Asn Val Lys Gly Val Tyr 185 190 195 gta gat gaa gat gat gct gga ata tac ata gtc aaa tca att gat gat 799 Val Asp Glu Asp Asp Ala Gly Ile Tyr Ile Val Lys Ser Ile Asp Asp 200 205 210 cct cgc aca ctc aac aag act gtg tat atc agg cca cca atg aat ata 847 Pro Arg Thr Leu Asn Lys Thr Val Tyr Ile Arg Pro Pro Met Asn Ile 215 220 225 ctt tca cag aaa gaa gta gtt gaa ata tgg gag aga cta tca ggt ttg 895 Leu Ser Gln Lys Glu Val Val Glu Ile Trp Glu Arg Leu Ser Gly Leu 230 235 240 agc cta gaa aaa atc tac gtt tct gag gac caa ctt ctt aat atg aaa 943 Ser Leu Glu Lys Ile Tyr Val Ser Glu Asp Gln Leu Leu Asn Met Lys 245 250 255 260 gat aaa tct tat gtg gag aag atg gca cga tgt cat ctc tat cat ttt 991 Asp Lys Ser Tyr Val Glu Lys Met Ala Arg Cys His Leu Tyr His Phe 265 270 275 ttt atc aaa ggg gat ctt tac aat ttt gaa att gga ccc aat gct act 1039 Phe Ile Lys Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asn Ala Thr 280 285 290 gaa ggc aca aaa ctt tat cca gaa gtc aaa tac aca acc atg gat tca 1087 Glu Gly Thr Lys Leu Tyr Pro Glu Val Lys Tyr Thr Thr Met Asp Ser 295 300 305 tat atg gag cgt tat cta tagctaatag atttttctta aataatagct 1135 Tyr Met Glu Arg Tyr Leu 310 tgaaatattc tatactcaat aagagtgtat tcataaataa tacacaacac ttgctctttt 1195 atagattact tttttaatag gtggctttta taaaacatgt ataaaaaaaa ttgcaaacaa 1255 tatttttaaa ttagcaataa taaccacctt taaataaaaa aaaaaaaaaa aaa 1308 66 314 PRT Thuja plicata 66 Met Asp Lys Lys Ser Arg Val Leu Ile Val Gly Gly Thr Gly Phe Ile 1 5 10 15 Gly Lys Arg Ile Val Lys Ala Ser Leu Ala Leu Gly His Pro Thr Tyr 20 25 30 Val Leu Phe Arg Pro Glu Ala Leu Ser Tyr Ile Asp Lys Val Gln Met 35 40 45 Leu Ile Ser Phe Lys Gln Leu Gly Ala Lys Leu Leu Glu Ala Ser Leu 50 55 60 Asp Asp His Gln Gly Leu Val Asp Val Val Lys Gln Val Asp Val Val 65 70 75 80 Ile Ser Ala Val Ser Gly Gly Leu Val Arg His His Ile Leu Asp Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe 100 105 110 Leu Pro Ser Glu Phe Gly Met Asp Pro Asp Val Val Glu Asp Pro Leu 115 120 125 Glu Pro Gly Asn Ile Thr Phe Ile Asp Lys Arg Lys Val Arg Arg Ala 130 135 140 Ile Glu Ala Ala Thr Ile Pro Tyr Thr Tyr Val Ser Ser Asn Met Phe 145 150 155 160 Ala Gly Phe Phe Ala Gly Ser Leu Ala Gln Leu Gln Asp Ala Pro Arg 165 170 175 Met Met Pro Ala Arg Asp Lys Val Leu Ile Tyr Gly Asp Gly Asn Val 180 185 190 Lys Gly Val Tyr Val Asp Glu Asp Asp Ala Gly Ile Tyr Ile Val Lys 195 200 205 Ser Ile Asp Asp Pro Arg Thr Leu Asn Lys Thr Val Tyr Ile Arg Pro 210 215 220 Pro Met Asn Ile Leu Ser Gln Lys Glu Val Val Glu Ile Trp Glu Arg 225 230 235 240 Leu Ser Gly Leu Ser Leu Glu Lys Ile Tyr Val Ser Glu Asp Gln Leu 245 250 255 Leu Asn Met Lys Asp Lys Ser Tyr Val Glu Lys Met Ala Arg Cys His 260 265 270 Leu Tyr His Phe Phe Ile Lys Gly Asp Leu Tyr Asn Phe Glu Ile Gly 275 280 285 Pro Asn Ala Thr Glu Gly Thr Lys Leu Tyr Pro Glu Val Lys Tyr Thr 290 295 300 Thr Met Asp Ser Tyr Met Glu Arg Tyr Leu 305 310 67 1287 DNA Thuja plicata CDS (11)..(946) 67 gaaagcagag atg gaa gag agt agc agg att ttg gta gtg gga ggc aca 49 Met Glu Glu Ser Ser Arg Ile Leu Val Val Gly Gly Thr 1 5 10 gga tac ata ggc aga agg att gtg aaa gcc agc att gct ctg ggc cat 97 Gly Tyr Ile Gly Arg Arg Ile Val Lys Ala Ser Ile Ala Leu Gly His 15 20 25 cct act ttc att ttg ttt agg aaa gaa gtt gtt tct gat gta gag aaa 145 Pro Thr Phe Ile Leu Phe Arg Lys Glu Val Val Ser Asp Val Glu Lys 30 35 40 45 gtg gag atg tta ttg tcc ttc aaa aag aat ggt gcc aaa tta ctg gag 193 Val Glu Met Leu Leu Ser Phe Lys Lys Asn Gly Ala Lys Leu Leu Glu 50 55 60 gct tca ttt gat gat cac gaa agc ctt gta gat gct gtg aag cag gtt 241 Ala Ser Phe Asp Asp His Glu Ser Leu Val Asp Ala Val Lys Gln Val 65 70 75 gat gtt gtc ata agt gca gtt gca gga aac cac atg cgg cat cac atc 289 Asp Val Val Ile Ser Ala Val Ala Gly Asn His Met Arg His His Ile 80 85 90 ctt caa cag ctc aaa tta gtg gag gcc att aaa gaa gct gga aat att 337 Leu Gln Gln Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile 95 100 105 aag agg ttt gtc cct tca gaa ttt ggg atg gat cca ggg tta atg gac 385 Lys Arg Phe Val Pro Ser Glu Phe Gly Met Asp Pro Gly Leu Met Asp 110 115 120 125 cat gca atg gca cca gga aac att gta ttt att gat aaa ata aaa gtt 433 His Ala Met Ala Pro Gly Asn Ile Val Phe Ile Asp Lys Ile Lys Val 130 135 140 cga gag gcc att gaa gct gca gct att cct cac act tat att tct gcc 481 Arg Glu Ala Ile Glu Ala Ala Ala Ile Pro His Thr Tyr Ile Ser Ala 145 150 155 aat ata ttt gct ggc tac ttg gtt ggt gga tta gct caa ctt ggt cgt 529 Asn Ile Phe Ala Gly Tyr Leu Val Gly Gly Leu Ala Gln Leu Gly Arg 160 165 170 gtg atg cct cct tca gac aaa gta ttt ctc tat gga gat gga aat gtc 577 Val Met Pro Pro Ser Asp Lys Val Phe Leu Tyr Gly Asp Gly Asn Val 175 180 185 aaa gct gtt tgg ata gat gaa gaa gat gtt gga ata tac aca atc aaa 625 Lys Ala Val Trp Ile Asp Glu Glu Asp Val Gly Ile Tyr Thr Ile Lys 190 195 200 205 gca att gat gac cct cgc acc cta aat aag act gtg tac atc agg cca 673 Ala Ile Asp Asp Pro Arg Thr Leu Asn Lys Thr Val Tyr Ile Arg Pro 210 215 220 cct ttg aat gtt ctt tcc cag aag gaa gtg gtt gaa aaa tgg gaa aaa 721 Pro Leu Asn Val Leu Ser Gln Lys Glu Val Val Glu Lys Trp Glu Lys 225 230 235 tta tca aga aag agc ttg gat aaa ata tat atg tct gtt gag gat ttt 769 Leu Ser Arg Lys Ser Leu Asp Lys Ile Tyr Met Ser Val Glu Asp Phe 240 245 250 ctc gca ggc atg gaa ggt caa tca tat gga gag aag att gga ata tca 817 Leu Ala Gly Met Glu Gly Gln Ser Tyr Gly Glu Lys Ile Gly Ile Ser 255 260 265 cat ttc tat cag atg ttc tat aag ggg gat ctt tat aat ttt gaa att 865 His Phe Tyr Gln Met Phe Tyr Lys Gly Asp Leu Tyr Asn Phe Glu Ile 270 275 280 285 gga cct aat gga gta gaa gct tcc caa ctt tac cca gga gta aaa tac 913 Gly Pro Asn Gly Val Glu Ala Ser Gln Leu Tyr Pro Gly Val Lys Tyr 290 295 300 aca aca gtg gac tca tac atg gag cgc tac cta tgaaaatctt cttcatgaag 966 Thr Thr Val Asp Ser Tyr Met Glu Arg Tyr Leu 305 310 atatttaaat tcaatttaat gctttctaaa agtttttata ttttgacata atgctaaata 1026 tagatgtaga gtatctagat aataatattc aattgataat attcaacaat cagttgagat 1086 gactttttcc ctttaactgc atgctcaaca tattttatac aaacaagcta atgtctttta 1146 aggttgagaa actaaatatg gttttgtatt acatggaaaa accatatttt gatatttgag 1206 attgtattta ttttgaatgt tatgattttg ataaaatttg aaattgatta tgaacattgt 1266 tttaaaaaaa aaaaaaaaaa a 1287 68 312 PRT Thuja plicata 68 Met Glu Glu Ser Ser Arg Ile Leu Val Val Gly Gly Thr Gly Tyr Ile 1 5 10 15 Gly Arg Arg Ile Val Lys Ala Ser Ile Ala Leu Gly His Pro Thr Phe 20 25 30 Ile Leu Phe Arg Lys Glu Val Val Ser Asp Val Glu Lys Val Glu Met 35 40 45 Leu Leu Ser Phe Lys Lys Asn Gly Ala Lys Leu Leu Glu Ala Ser Phe 50 55 60 Asp Asp His Glu Ser Leu Val Asp Ala Val Lys Gln Val Asp Val Val 65 70 75 80 Ile Ser Ala Val Ala Gly Asn His Met Arg His His Ile Leu Gln Gln 85 90 95 Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe 100 105 110 Val Pro Ser Glu Phe Gly Met Asp Pro Gly Leu Met Asp His Ala Met 115 120 125 Ala Pro Gly Asn Ile Val Phe Ile Asp Lys Ile Lys Val Arg Glu Ala 130 135 140 Ile Glu Ala Ala Ala Ile Pro His Thr Tyr Ile Ser Ala Asn Ile Phe 145 150 155 160 Ala Gly Tyr Leu Val Gly Gly Leu Ala Gln Leu Gly Arg Val Met Pro 165 170 175 Pro Ser Asp Lys Val Phe Leu Tyr Gly Asp Gly Asn Val Lys Ala Val 180 185 190 Trp Ile Asp Glu Glu Asp Val Gly Ile Tyr Thr Ile Lys Ala Ile Asp 195 200 205 Asp Pro Arg Thr Leu Asn Lys Thr Val Tyr Ile Arg Pro Pro Leu Asn 210 215 220 Val Leu Ser Gln Lys Glu Val Val Glu Lys Trp Glu Lys Leu Ser Arg 225 230 235 240 Lys Ser Leu Asp Lys Ile Tyr Met Ser Val Glu Asp Phe Leu Ala Gly 245 250 255 Met Glu Gly Gln Ser Tyr Gly Glu Lys Ile Gly Ile Ser His Phe Tyr 260 265 270 Gln Met Phe Tyr Lys Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asn 275 280 285 Gly Val Glu Ala Ser Gln Leu Tyr Pro Gly Val Lys Tyr Thr Thr Val 290 295 300 Asp Ser Tyr Met Glu Arg Tyr Leu 305 310 69 1282 DNA Tsuga heterophylla CDS (2)..(922) 69 c aga gtt cta ata gtg ggt ggc aca gga tac ata ggt aga aaa ttt gta 49 Arg Val Leu Ile Val Gly Gly Thr Gly Tyr Ile Gly Arg Lys Phe Val 1 5 10 15 aaa gct agc tta gct cta ggc cac cca aca ttc gtt ttg tcc agg cca 97 Lys Ala Ser Leu Ala Leu Gly His Pro Thr Phe Val Leu Ser Arg Pro 20 25 30 gaa gta ggg ttt gac att gag aag gtg cac atg ttg ctc tcc ttc aaa 145 Glu Val Gly Phe Asp Ile Glu Lys Val His Met Leu Leu Ser Phe Lys 35 40 45 caa gcg ggt gcc aga ctt ttg gag ggt tca ttt gag gat ttc caa agc 193 Gln Ala Gly Ala Arg Leu Leu Glu Gly Ser Phe Glu Asp Phe Gln Ser 50 55 60 ctt gtg gca gcc ttg aag cag gtt gat gtt gtg ata agt gca gtg gca 241 Leu Val Ala Ala Leu Lys Gln Val Asp Val Val Ile Ser Ala Val Ala 65 70 75 80 gga aac cat ttc aga aac ctt ata ctt caa cag ctt aaa ttg gtg gaa 289 Gly Asn His Phe Arg Asn Leu Ile Leu Gln Gln Leu Lys Leu Val Glu 85 90 95 gcc ata aaa gaa gct ggc aac att aag aga ttt ctt cct tct gaa ttt 337 Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe Leu Pro Ser Glu Phe 100 105 110 gga atg gaa cca gac ctc atg gag cac gct ttg gaa cct ggt aac gct 385 Gly Met Glu Pro Asp Leu Met Glu His Ala Leu Glu Pro Gly Asn Ala 115 120 125 gtc ttc att gat aag aga aag gtt cgg cgc gcc att gaa gca gca ggc 433 Val Phe Ile Asp Lys Arg Lys Val Arg Arg Ala Ile Glu Ala Ala Gly 130 135 140 att cct tac acg tat gtc tct tca aat ata ttt gct ggg tat tta gca 481 Ile Pro Tyr Thr Tyr Val Ser Ser Asn Ile Phe Ala Gly Tyr Leu Ala 145 150 155 160 gga ggg ttg gca caa att ggc cgg ctt atg cct cct cgt gat gaa gta 529 Gly Gly Leu Ala Gln Ile Gly Arg Leu Met Pro Pro Arg Asp Glu Val 165 170 175 gtt atc tat gga gat ggt aac gtt aaa gct gtt tgg gtg gac gaa gat 577 Val Ile Tyr Gly Asp Gly Asn Val Lys Ala Val Trp Val Asp Glu Asp 180 185 190 gat gtc gga ata tac aca ctg aaa aca atc gat gat cca cgc act ctg 625 Asp Val Gly Ile Tyr Thr Leu Lys Thr Ile Asp Asp Pro Arg Thr Leu 195 200 205 aac aag act gta tat atc agg cca ctc aaa aat att ctc tct cag aag 673 Asn Lys Thr Val Tyr Ile Arg Pro Leu Lys Asn Ile Leu Ser Gln Lys 210 215 220 gag ctt gtg gca aag tgg gaa aaa ctc tca gga aag tgt ttg aag aaa 721 Glu Leu Val Ala Lys Trp Glu Lys Leu Ser Gly Lys Cys Leu Lys Lys 225 230 235 240 aca tac att tct gct gag gat ttt ctt gca ggc atc gaa gat caa cct 769 Thr Tyr Ile Ser Ala Glu Asp Phe Leu Ala Gly Ile Glu Asp Gln Pro 245 250 255 tac gaa cat cag gtc gga ata tct cac ttc tat caa atg ttt tac agt 817 Tyr Glu His Gln Val Gly Ile Ser His Phe Tyr Gln Met Phe Tyr Ser 260 265 270 gga gat ctc tat aat ttt gag att ggg cca gac ggt aga gaa gca aca 865 Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asp Gly Arg Glu Ala Thr 275 280 285 gtg cta tac cct gaa gtt caa tac act acc atg gat tct tat ttg aag 913 Val Leu Tyr Pro Glu Val Gln Tyr Thr Thr Met Asp Ser Tyr Leu Lys 290 295 300 cgc tac tta taagcaggat gaaggttaat gttctacgac atgaatccca 962 Arg Tyr Leu 305 cgagaaatac cagaaatctt cattcaagat caaataatgg ataaataatt caacattagt 1022 tccatcagaa ataccagaaa tttctaatcg agttcaaata atggataaat aattcattat 1082 ttaagtttta tttatcgaaa tagggctgga cgaattgaat atatattcat ctgatatgga 1142 cgggcaggtt gtaaaattgc aagctgtaca gtaactacgt cttgtcgcga aaagctacta 1202 tatcgatata actgatgtga aaagttacca tttcgtaata actatgcttg aatttatttt 1262 tgacaaaaaa aaaaaaaaaa 1282 70 307 PRT Tsuga heterophylla 70 Arg Val Leu Ile Val Gly Gly Thr Gly Tyr Ile Gly Arg Lys Phe Val 1 5 10 15 Lys Ala Ser Leu Ala Leu Gly His Pro Thr Phe Val Leu Ser Arg Pro 20 25 30 Glu Val Gly Phe Asp Ile Glu Lys Val His Met Leu Leu Ser Phe Lys 35 40 45 Gln Ala Gly Ala Arg Leu Leu Glu Gly Ser Phe Glu Asp Phe Gln Ser 50 55 60 Leu Val Ala Ala Leu Lys Gln Val Asp Val Val Ile Ser Ala Val Ala 65 70 75 80 Gly Asn His Phe Arg Asn Leu Ile Leu Gln Gln Leu Lys Leu Val Glu 85 90 95 Ala Ile Lys Glu Ala Gly Asn Ile Lys Arg Phe Leu Pro Ser Glu Phe 100 105 110 Gly Met Glu Pro Asp Leu Met Glu His Ala Leu Glu Pro Gly Asn Ala 115 120 125 Val Phe Ile Asp Lys Arg Lys Val Arg Arg Ala Ile Glu Ala Ala Gly 130 135 140 Ile Pro Tyr Thr Tyr Val Ser Ser Asn Ile Phe Ala Gly Tyr Leu Ala 145 150 155 160 Gly Gly Leu Ala Gln Ile Gly Arg Leu Met Pro Pro Arg Asp Glu Val 165 170 175 Val Ile Tyr Gly Asp Gly Asn Val Lys Ala Val Trp Val Asp Glu Asp 180 185 190 Asp Val Gly Ile Tyr Thr Leu Lys Thr Ile Asp Asp Pro Arg Thr Leu 195 200 205 Asn Lys Thr Val Tyr Ile Arg Pro Leu Lys Asn Ile Leu Ser Gln Lys 210 215 220 Glu Leu Val Ala Lys Trp Glu Lys Leu Ser Gly Lys Cys Leu Lys Lys 225 230 235 240 Thr Tyr Ile Ser Ala Glu Asp Phe Leu Ala Gly Ile Glu Asp Gln Pro 245 250 255 Tyr Glu His Gln Val Gly Ile Ser His Phe Tyr Gln Met Phe Tyr Ser 260 265 270 Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asp Gly Arg Glu Ala Thr 275 280 285 Val Leu Tyr Pro Glu Val Gln Tyr Thr Thr Met Asp Ser Tyr Leu Lys 290 295 300 Arg Tyr Leu 305 71 1328 DNA Tsuga heterophylla CDS (20)..(946) 71 gaattcggca cgagctaac atg agc aga gtt cta ata gtg ggt ggc aca gga 52 Met Ser Arg Val Leu Ile Val Gly Gly Thr Gly 1 5 10 tac ata ggt aga aaa ttt gta aaa gct agc tta gct cta ggc cac cca 100 Tyr Ile Gly Arg Lys Phe Val Lys Ala Ser Leu Ala Leu Gly His Pro 15 20 25 aca ttc gtt ttg tcc agg cca gaa gta ggg ttt gac att gag aag gtg 148 Thr Phe Val Leu Ser Arg Pro Glu Val Gly Phe Asp Ile Glu Lys Val 30 35 40 cac atg ttg ctc tcc ttc aaa caa gcg ggt gcc aga ctt ttg gag ggt 196 His Met Leu Leu Ser Phe Lys Gln Ala Gly Ala Arg Leu Leu Glu Gly 45 50 55 tca ttt gag gat ttc caa agc ctt gtg gca gcc ttg aag cag gtt gat 244 Ser Phe Glu Asp Phe Gln Ser Leu Val Ala Ala Leu Lys Gln Val Asp 60 65 70 75 gtt gtg ata agt gca gtg gca gga aac cat ttc aga aac ctt ata ctt 292 Val Val Ile Ser Ala Val Ala Gly Asn His Phe Arg Asn Leu Ile Leu 80 85 90 caa cag ctt aaa ttg gtg gaa gcc ata aaa gag gct cgc aac att aag 340 Gln Gln Leu Lys Leu Val Glu Ala Ile Lys Glu Ala Arg Asn Ile Lys 95 100 105 aga ttt ctt cct tct gaa ttt gga atg gac cca gac ctc atg gag cac 388 Arg Phe Leu Pro Ser Glu Phe Gly Met Asp Pro Asp Leu Met Glu His 110 115 120 gct ttg gaa cct ggt aac gct gtc ttc att gat aag aga aag gtt cgg 436 Ala Leu Glu Pro Gly Asn Ala Val Phe Ile Asp Lys Arg Lys Val Arg 125 130 135 cgc gcc att gaa gca gca ggc att cct tac acg tat gtc tct tca aat 484 Arg Ala Ile Glu Ala Ala Gly Ile Pro Tyr Thr Tyr Val Ser Ser Asn 140 145 150 155 ata ttt gct ggg tat tta gca gga ggg ttg gca caa att ggc cgg ctt 532 Ile Phe Ala Gly Tyr Leu Ala Gly Gly Leu Ala Gln Ile Gly Arg Leu 160 165 170 atg cct cct cgt gat gaa gta gtt atc tat gga gat ggt aac gtt aaa 580 Met Pro Pro Arg Asp Glu Val Val Ile Tyr Gly Asp Gly Asn Val Lys 175 180 185 gct gtt tgg gtg gac gaa gat gat gtc gga ata tac aca ctg aaa aca 628 Ala Val Trp Val Asp Glu Asp Asp Val Gly Ile Tyr Thr Leu Lys Thr 190 195 200 atc gat gat cca cgc act ctg aac aag act gta tat atc agg cca ctc 676 Ile Asp Asp Pro Arg Thr Leu Asn Lys Thr Val Tyr Ile Arg Pro Leu 205 210 215 aaa aat ata ctc tct cag aag gag ctt gtg gca aag tgg gaa aaa ctc 724 Lys Asn Ile Leu Ser Gln Lys Glu Leu Val Ala Lys Trp Glu Lys Leu 220 225 230 235 tca gga aag ttt ttg aag aaa aca tac att tct gct gag gat ttt ctt 772 Ser Gly Lys Phe Leu Lys Lys Thr Tyr Ile Ser Ala Glu Asp Phe Leu 240 245 250 gca ggc atc gaa gat caa cct tac gaa cat cag gtc gga ata tct cac 820 Ala Gly Ile Glu Asp Gln Pro Tyr Glu His Gln Val Gly Ile Ser His 255 260 265 ttc tat caa atg ttt tac agt gga gat ctc tat aat ttt gag att ggg 868 Phe Tyr Gln Met Phe Tyr Ser Gly Asp Leu Tyr Asn Phe Glu Ile Gly 270 275 280 cca gac ggt aga gaa gca aca atg cta tac cct gaa gtt caa tac act 916 Pro Asp Gly Arg Glu Ala Thr Met Leu Tyr Pro Glu Val Gln Tyr Thr 285 290 295 acc atg gat tct tat ttg aag cgc tac tta taagcaggat gaaggttaat 966 Thr Met Asp Ser Tyr Leu Lys Arg Tyr Leu 300 305 gttctacgac atgaatccca cgagaaatac cagaaatctt cattcaagat caaataatgg 1026 ataaataatt caacattagt tccatcagaa atatcagaaa tttctaatca agttcaaata 1086 atggataaat aattcattat ttaagtttta tttattgaaa tagggctgga cgaagccttt 1146 aatcagtatt gaatatatat tcatctgata tggacgggca ggttgtaaaa ttgcaagccg 1206 tacagtaact acgtcttgtc gcgaaaagct accatatcga tataactaag tcttgtcgcg 1266 taaagctacc atatcgatat aactgatgtg accatttcgt aataactatg cttgtgcagg 1326 aa 1328 72 309 PRT Tsuga heterophylla 72 Met Ser Arg Val Leu Ile Val Gly Gly Thr Gly Tyr Ile Gly Arg Lys 1 5 10 15 Phe Val Lys Ala Ser Leu Ala Leu Gly His Pro Thr Phe Val Leu Ser 20 25 30 Arg Pro Glu Val Gly Phe Asp Ile Glu Lys Val His Met Leu Leu Ser 35 40 45 Phe Lys Gln Ala Gly Ala Arg Leu Leu Glu Gly Ser Phe Glu Asp Phe 50 55 60 Gln Ser Leu Val Ala Ala Leu Lys Gln Val Asp Val Val Ile Ser Ala 65 70 75 80 Val Ala Gly Asn His Phe Arg Asn Leu Ile Leu Gln Gln Leu Lys Leu 85 90 95 Val Glu Ala Ile Lys Glu Ala Arg Asn Ile Lys Arg Phe Leu Pro Ser 100 105 110 Glu Phe Gly Met Asp Pro Asp Leu Met Glu His Ala Leu Glu Pro Gly 115 120 125 Asn Ala Val Phe Ile Asp Lys Arg Lys Val Arg Arg Ala Ile Glu Ala 130 135 140 Ala Gly Ile Pro Tyr Thr Tyr Val Ser Ser Asn Ile Phe Ala Gly Tyr 145 150 155 160 Leu Ala Gly Gly Leu Ala Gln Ile Gly Arg Leu Met Pro Pro Arg Asp 165 170 175 Glu Val Val Ile Tyr Gly Asp Gly Asn Val Lys Ala Val Trp Val Asp 180 185 190 Glu Asp Asp Val Gly Ile Tyr Thr Leu Lys Thr Ile Asp Asp Pro Arg 195 200 205 Thr Leu Asn Lys Thr Val Tyr Ile Arg Pro Leu Lys Asn Ile Leu Ser 210 215 220 Gln Lys Glu Leu Val Ala Lys Trp Glu Lys Leu Ser Gly Lys Phe Leu 225 230 235 240 Lys Lys Thr Tyr Ile Ser Ala Glu Asp Phe Leu Ala Gly Ile Glu Asp 245 250 255 Gln Pro Tyr Glu His Gln Val Gly Ile Ser His Phe Tyr Gln Met Phe 260 265 270 Tyr Ser Gly Asp Leu Tyr Asn Phe Glu Ile Gly Pro Asp Gly Arg Glu 275 280 285 Ala Thr Met Leu Tyr Pro Glu Val Gln Tyr Thr Thr Met Asp Ser Tyr 290 295 300 Leu Lys Arg Tyr Leu 305 73 355 DNA Forsythia x intermedia 73 aaggagctgg tgttctactt ccacgacata cttttcaaag gggataatta caacaatgcc 60 actgccacca tagtcgggtc cccccaatgg ggcaacaaga ctgccatggc cgtgccattc 120 aattttggtg acctaatggt gttcgacgat cccattacct tagacaacaa tctgcattca 180 cccccagtgg gtcgggcaca agggatgtac ttctatgatc aaaaaagtac atacaatgct 240 tggctcgggt tctcattttt gttcaattca actaagtatg ttggaacctt gaactttgct 300 ggggctgatc cattgttgaa caagactagg gacgtatcag tcattggtgg aacca 355 74 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 74 cagctatgac catgattacg 20 75 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 75 gttttcccag tcacgacgt 19 76 6 PRT Artificial Sequence Description of Artificial Sequence conserved domain 76 Gly Xaa Gly Xaa Xaa Gly 1 5 77 582 DNA Thuja plicata CDS (1)..(582) 77 atg gca atc tgg aat ggt aga gtt ctg aac ttg tgc att ctg tgg ctt 48 Met Ala Ile Trp Asn Gly Arg Val Leu Asn Leu Cys Ile Leu Trp Leu 1 5 10 15 ctg gtc tcc aca gtt ttg ctg aat gat gca gat tgc cat agc tgg aaa 96 Leu Val Ser Thr Val Leu Leu Asn Asp Ala Asp Cys His Ser Trp Lys 20 25 30 aag aag ctt cca aag ccc cgt aag aat ctt gtt ttg tat ttc cat gac 144 Lys Lys Leu Pro Lys Pro Arg Lys Asn Leu Val Leu Tyr Phe His Asp 35 40 45 ata atc tac aat ggg caa aat gca gag aat gca act tct aca att gtt 192 Ile Ile Tyr Asn Gly Gln Asn Ala Glu Asn Ala Thr Ser Thr Ile Val 50 55 60 gca gcc cct gaa gga gcc aat ctc act att ttg act ggc aac aac cat 240 Ala Ala Pro Glu Gly Ala Asn Leu Thr Ile Leu Thr Gly Asn Asn His 65 70 75 80 ttt ggg aat att gct gtg ttt gat gat cct att act ctt gac aac aat 288 Phe Gly Asn Ile Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 ctt cat tct cct cca gtg ggt aga cct cag ggc ttt tac ttc tat gac 336 Leu His Ser Pro Pro Val Gly Arg Pro Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 atg aag aat aca ttc agt tct tgg ctt ggc ttc aca ttt gtg ctg aat 384 Met Lys Asn Thr Phe Ser Ser Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 tca acg gac tat aag ggc acc att act ttc aat gga gca gac cca att 432 Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 ttg gtt aag tac aga gat ata tct gtt gtg ggt gga acg ggg gat ttg 480 Leu Val Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Leu 145 150 155 160 tta atg gcc aga gga att gct gca atc aat act gat gca tat gag gga 528 Leu Met Ala Arg Gly Ile Ala Ala Ile Asn Thr Asp Ala Tyr Glu Gly 165 170 175 gat gtt tat ttc cgt ctt aga gtg aat att aca ctg tat gag tgc tac 576 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 tac tga 582 Tyr 78 193 PRT Thuja plicata 78 Met Ala Ile Trp Asn Gly Arg Val Leu Asn Leu Cys Ile Leu Trp Leu 1 5 10 15 Leu Val Ser Thr Val Leu Leu Asn Asp Ala Asp Cys His Ser Trp Lys 20 25 30 Lys Lys Leu Pro Lys Pro Arg Lys Asn Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Ile Tyr Asn Gly Gln Asn Ala Glu Asn Ala Thr Ser Thr Ile Val 50 55 60 Ala Ala Pro Glu Gly Ala Asn Leu Thr Ile Leu Thr Gly Asn Asn His 65 70 75 80 Phe Gly Asn Ile Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 Leu His Ser Pro Pro Val Gly Arg Pro Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asn Thr Phe Ser Ser Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asp Tyr Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 Leu Val Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Leu 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Ala Ile Asn Thr Asp Ala Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 Tyr 79 25 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 79 agagtggaga ttgttgtcaa gagta 25 80 39 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 80 gaccacgcgt atcgatgtcg actttttttt ttttttttv 39 81 22 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 81 gaccacgcgt atcgatgtcg ac 22 82 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 82 agtaatagga tcatcaaaca c 21 83 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 83 ccaacttctt tctctacttc agaa 24 84 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 84 cagaaccctg ttttctgatt tatt 24 85 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 85 tttatttttg cacaatggca atct 24 86 576 DNA Thuja plicata CDS (1)..(576) 86 atg gca atg aaa gct gct aga gtt ctg cat tta tgc ttt cta tgg ctt 48 Met Ala Met Lys Ala Ala Arg Val Leu His Leu Cys Phe Leu Trp Leu 1 5 10 15 cta gta tct gca atc ttc ata aaa tct gca gat tgc cgt agc tgg aaa 96 Leu Val Ser Ala Ile Phe Ile Lys Ser Ala Asp Cys Arg Ser Trp Lys 20 25 30 aag aag ctt cca aag ccc tgt aga aat ctt gtg tta tat ttt cat gat 144 Lys Lys Leu Pro Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp 35 40 45 ata atc tac aat ggc aaa aat gca gag aat gca aca tct gca ctt gtt 192 Ile Ile Tyr Asn Gly Lys Asn Ala Glu Asn Ala Thr Ser Ala Leu Val 50 55 60 tca gcc cct caa gga gct aat ctc acc att atg act ggt aat aac cat 240 Ser Ala Pro Gln Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His 65 70 75 80 ttt ggg aat ctt gca gtg ttt gat gat cct att act ctt gac aac aat 288 Phe Gly Asn Leu Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 ctt cac tct cct cct gtt gga aga gct cag ggc ttt tac ttc tat gac 336 Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 atg aag aac acc ttc agt gcc tgg ctt ggc ttc aca ttt gtg ctc aat 384 Met Lys Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 tca act gat cac aag ggc tcc att act ttc aat gga gca gat ccc atc 432 Ser Thr Asp His Lys Gly Ser Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 tta aca aag tac aga gac ata tct gtt gtg ggt gga aca ggg gat ttc 480 Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 ttg atg gca aga gga att gct acc att tct act gac tca tat gag gga 528 Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly 165 170 175 gat gtt tat ttc agg ctt agg gtc aat atc aca ctc tat gag tgt tac 576 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 87 192 PRT Thuja plicata 87 Met Ala Met Lys Ala Ala Arg Val Leu His Leu Cys Phe Leu Trp Leu 1 5 10 15 Leu Val Ser Ala Ile Phe Ile Lys Ser Ala Asp Cys Arg Ser Trp Lys 20 25 30 Lys Lys Leu Pro Lys Pro Cys Arg Asn Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Ile Tyr Asn Gly Lys Asn Ala Glu Asn Ala Thr Ser Ala Leu Val 50 55 60 Ser Ala Pro Gln Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His 65 70 75 80 Phe Gly Asn Leu Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asn Thr Phe Ser Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asp His Lys Gly Ser Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 88 22 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 88 gtaatacgac tcactatagg gc 22 89 28 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 89 agattagctc cttgaggggc tgaaacaa 28 90 19 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 90 actatagggc acgcgtggt 19 91 28 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 91 aggggctgaa acaagtgcag atgttgca 28 92 29 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 92 caagtgtgca gatgttgcat tctctgcat 29 93 576 DNA Thuja plicata CDS (1)..(576) 93 atg gca atg aaa tct gaa aga gct ttg cag tta tgc ctt ctg tgg ctt 48 Met Ala Met Lys Ser Glu Arg Ala Leu Gln Leu Cys Leu Leu Trp Leu 1 5 10 15 ctg atg tct gca atc ttg cta aaa cct gca gat tgc cat gga agg aag 96 Leu Met Ser Ala Ile Leu Leu Lys Pro Ala Asp Cys His Gly Arg Lys 20 25 30 aag agg ctt ccc aag ccc tgc aag cat ctt gtg ttg tat ttc cat gat 144 Lys Arg Leu Pro Lys Pro Cys Lys His Leu Val Leu Tyr Phe His Asp 35 40 45 ata ctc tac aat ggc aag aat gcc cac aat gca aca tct gca ctt gtt 192 Ile Leu Tyr Asn Gly Lys Asn Ala His Asn Ala Thr Ser Ala Leu Val 50 55 60 gca gcc cct gag gga gcc aat ctc acc att atg act ggt aat aac cat 240 Ala Ala Pro Glu Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His 65 70 75 80 ttt ggg aat att gct gtg ttt gat gat cct att act ctt gac aac aat 288 Phe Gly Asn Ile Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 ctt cac tct cct tct gtt gga aga gct cag ggc ttt tac ttc tat gac 336 Leu His Ser Pro Ser Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 atg aag gat acc ttc aat gct tgg ctt ggt ttt aca ttt gtg ctg aat 384 Met Lys Asp Thr Phe Asn Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 tca act gat cac aag ggc acc att act ttc aat gga gca gat cca atc 432 Ser Thr Asp His Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 ctg acc aag tac aga gat ata tct gtt gtg ggt gga aca ggg gat ttc 480 Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 ttg atg gcc aga gga att gcc acc att tct act gat tca tat gag gga 528 Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly 165 170 175 gat gtt tat ttc agg ctt agg gtc aat atc aca ctc tat gag tgt tac 576 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 94 192 PRT Thuja plicata 94 Met Ala Met Lys Ser Glu Arg Ala Leu Gln Leu Cys Leu Leu Trp Leu 1 5 10 15 Leu Met Ser Ala Ile Leu Leu Lys Pro Ala Asp Cys His Gly Arg Lys 20 25 30 Lys Arg Leu Pro Lys Pro Cys Lys His Leu Val Leu Tyr Phe His Asp 35 40 45 Ile Leu Tyr Asn Gly Lys Asn Ala His Asn Ala Thr Ser Ala Leu Val 50 55 60 Ala Ala Pro Glu Gly Ala Asn Leu Thr Ile Met Thr Gly Asn Asn His 65 70 75 80 Phe Gly Asn Ile Ala Val Phe Asp Asp Pro Ile Thr Leu Asp Asn Asn 85 90 95 Leu His Ser Pro Ser Val Gly Arg Ala Gln Gly Phe Tyr Phe Tyr Asp 100 105 110 Met Lys Asp Thr Phe Asn Ala Trp Leu Gly Phe Thr Phe Val Leu Asn 115 120 125 Ser Thr Asp His Lys Gly Thr Ile Thr Phe Asn Gly Ala Asp Pro Ile 130 135 140 Leu Thr Lys Tyr Arg Asp Ile Ser Val Val Gly Gly Thr Gly Asp Phe 145 150 155 160 Leu Met Ala Arg Gly Ile Ala Thr Ile Ser Thr Asp Ser Tyr Glu Gly 165 170 175 Asp Val Tyr Phe Arg Leu Arg Val Asn Ile Thr Leu Tyr Glu Cys Tyr 180 185 190 95 27 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 95 cagtcataat ggtgagattg gctccct 27 96 27 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 96 tgagattggc tccctcaggg gctgcaa 27 97 27 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 97 ggctgcaaca agtgcagatg ttgcatt 27 98 552 DNA Eucommia ulmoides CDS (1)..(552) 98 atg gca aac ctt gtt gag aaa tct tat tac att atc ttc atg ctt gtt 48 Met Ala Asn Leu Val Glu Lys Ser Tyr Tyr Ile Ile Phe Met Leu Val 1 5 10 15 cta aca tca tcc tat gtt gtt gtc tcc tcc aag tcc aag aca atc cga 96 Leu Thr Ser Ser Tyr Val Val Val Ser Ser Lys Ser Lys Thr Ile Arg 20 25 30 ccc gaa aac cca tgc aac cgt atc gtc ctc tac tac cac gac atc ctc 144 Pro Glu Asn Pro Cys Asn Arg Ile Val Leu Tyr Tyr His Asp Ile Leu 35 40 45 ttc aac ggc acc aac acc gtt aat gcc aca tca gca aaa gcc gcc aaa 192 Phe Asn Gly Thr Asn Thr Val Asn Ala Thr Ser Ala Lys Ala Ala Lys 50 55 60 gag acc cgc ctc ggg tcc cac gaa ttt ggg atg ctc gtg gtt ttt gac 240 Glu Thr Arg Leu Gly Ser His Glu Phe Gly Met Leu Val Val Phe Asp 65 70 75 80 gat ccg gtg acg gca gac cgc gag ctc cag tcg cct ccg ttg ggc cgg 288 Asp Pro Val Thr Ala Asp Arg Glu Leu Gln Ser Pro Pro Leu Gly Arg 85 90 95 gct cag ggg ttc tac ttt tat gat atg aag agc gag tac aat gct tgg 336 Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Ser Glu Tyr Asn Ala Trp 100 105 110 ttt gca tat acg ttg gtg ttt aac tcg agc gag cat aaa ggg acg atc 384 Phe Ala Tyr Thr Leu Val Phe Asn Ser Ser Glu His Lys Gly Thr Ile 115 120 125 acg ata atg ggg gcc gat atg atg ggg gag aaa aca cgg gat ctt ccg 432 Thr Ile Met Gly Ala Asp Met Met Gly Glu Lys Thr Arg Asp Leu Pro 130 135 140 gtg gtt gga gga acg ggg gat ttt ttc atg gca aga ggg att gcc acg 480 Val Val Gly Gly Thr Gly Asp Phe Phe Met Ala Arg Gly Ile Ala Thr 145 150 155 160 ttt cga acc gat gct ttt gag ggg ttc aat tat ttt cgg ctt gag atg 528 Phe Arg Thr Asp Ala Phe Glu Gly Phe Asn Tyr Phe Arg Leu Glu Met 165 170 175 gat gtc aag ttg tac gag tgt tat 552 Asp Val Lys Leu Tyr Glu Cys Tyr 180 99 184 PRT Eucommia ulmoides 99 Met Ala Asn Leu Val Glu Lys Ser Tyr Tyr Ile Ile Phe Met Leu Val 1 5 10 15 Leu Thr Ser Ser Tyr Val Val Val Ser Ser Lys Ser Lys Thr Ile Arg 20 25 30 Pro Glu Asn Pro Cys Asn Arg Ile Val Leu Tyr Tyr His Asp Ile Leu 35 40 45 Phe Asn Gly Thr Asn Thr Val Asn Ala Thr Ser Ala Lys Ala Ala Lys 50 55 60 Glu Thr Arg Leu Gly Ser His Glu Phe Gly Met Leu Val Val Phe Asp 65 70 75 80 Asp Pro Val Thr Ala Asp Arg Glu Leu Gln Ser Pro Pro Leu Gly Arg 85 90 95 Ala Gln Gly Phe Tyr Phe Tyr Asp Met Lys Ser Glu Tyr Asn Ala Trp 100 105 110 Phe Ala Tyr Thr Leu Val Phe Asn Ser Ser Glu His Lys Gly Thr Ile 115 120 125 Thr Ile Met Gly Ala Asp Met Met Gly Glu Lys Thr Arg Asp Leu Pro 130 135 140 Val Val Gly Gly Thr Gly Asp Phe Phe Met Ala Arg Gly Ile Ala Thr 145 150 155 160 Phe Arg Thr Asp Ala Phe Glu Gly Phe Asn Tyr Phe Arg Leu Glu Met 165 170 175 Asp Val Lys Leu Tyr Glu Cys Tyr 180 100 28 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 100 garttggtgt tctatttcca cgacatmc 28 101 25 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 101 caaagtggca acccctgtcg ccatg 25 102 22 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 102 cccccgttcc tccaaccacc gg 22 103 26 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 103 ggcccatgcg gttaagcata ttctcc 26 104 29 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 104 cctctataaa aacataattc ttttccccc 29 105 588 DNA Schisandra chinensis CDS (1)..(588) 105 atg gaa ggg aga aag ctg atc atc act atc cct ctc ctc ctc ttc ttc 48 Met Glu Gly Arg Lys Leu Ile Ile Thr Ile Pro Leu Leu Leu Phe Phe 1 5 10 15 att gcc ttc ttc tca gtg cct ccg gct gcg ttt ggc cgg aaa gtg aca 96 Ile Ala Phe Phe Ser Val Pro Pro Ala Ala Phe Gly Arg Lys Val Thr 20 25 30 ctt ccc cgt aaa agg atg ccg caa cca tgc atg aac ttg gtg ttt tac 144 Leu Pro Arg Lys Arg Met Pro Gln Pro Cys Met Asn Leu Val Phe Tyr 35 40 45 ttc cac gac atc tta tac aac ggc aag aat gct gcc aat gca act tcg 192 Phe His Asp Ile Leu Tyr Asn Gly Lys Asn Ala Ala Asn Ala Thr Ser 50 55 60 gcg att gtc ggg tcg ccg gca tgg ggg aac cgg acc att cta gct gga 240 Ala Ile Val Gly Ser Pro Ala Trp Gly Asn Arg Thr Ile Leu Ala Gly 65 70 75 80 caa agc aat ttt ggt gac atg gtc gta ttt gat gac ccg att act ctt 288 Gln Ser Asn Phe Gly Asp Met Val Val Phe Asp Asp Pro Ile Thr Leu 85 90 95 gac aac aat ctg cat tcg ccc ccc gtt ggt cgt gcg cag gga ttc tac 336 Asp Asn Asn Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr 100 105 110 ttc tac gac agg aag gat gta ttt acc gcg tgg cta ggc ttc agt ttc 384 Phe Tyr Asp Arg Lys Asp Val Phe Thr Ala Trp Leu Gly Phe Ser Phe 115 120 125 gtc ttc aac aat tca gac tac agg ggg agt ata aat ttt gct ggc gca 432 Val Phe Asn Asn Ser Asp Tyr Arg Gly Ser Ile Asn Phe Ala Gly Ala 130 135 140 gat cca ctt ttg atc aag acg agg gac atc tct gtg atc ggt ggc acc 480 Asp Pro Leu Leu Ile Lys Thr Arg Asp Ile Ser Val Ile Gly Gly Thr 145 150 155 160 ggc gat ttt ttc atg gct aga ggg atc gcg aca ttg atg aca gat gcc 528 Gly Asp Phe Phe Met Ala Arg Gly Ile Ala Thr Leu Met Thr Asp Ala 165 170 175 ttc gag ggt gag gtg tat ttc agg ctg agg aca gat atc aag ctg tat 576 Phe Glu Gly Glu Val Tyr Phe Arg Leu Arg Thr Asp Ile Lys Leu Tyr 180 185 190 gaa tgc tac tga 588 Glu Cys Tyr 195 106 195 PRT Schisandra chinensis 106 Met Glu Gly Arg Lys Leu Ile Ile Thr Ile Pro Leu Leu Leu Phe Phe 1 5 10 15 Ile Ala Phe Phe Ser Val Pro Pro Ala Ala Phe Gly Arg Lys Val Thr 20 25 30 Leu Pro Arg Lys Arg Met Pro Gln Pro Cys Met Asn Leu Val Phe Tyr 35 40 45 Phe His Asp Ile Leu Tyr Asn Gly Lys Asn Ala Ala Asn Ala Thr Ser 50 55 60 Ala Ile Val Gly Ser Pro Ala Trp Gly Asn Arg Thr Ile Leu Ala Gly 65 70 75 80 Gln Ser Asn Phe Gly Asp Met Val Val Phe Asp Asp Pro Ile Thr Leu 85 90 95 Asp Asn Asn Leu His Ser Pro Pro Val Gly Arg Ala Gln Gly Phe Tyr 100 105 110 Phe Tyr Asp Arg Lys Asp Val Phe Thr Ala Trp Leu Gly Phe Ser Phe 115 120 125 Val Phe Asn Asn Ser Asp Tyr Arg Gly Ser Ile Asn Phe Ala Gly Ala 130 135 140 Asp Pro Leu Leu Ile Lys Thr Arg Asp Ile Ser Val Ile Gly Gly Thr 145 150 155 160 Gly Asp Phe Phe Met Ala Arg Gly Ile Ala Thr Leu Met Thr Asp Ala 165 170 175 Phe Glu Gly Glu Val Tyr Phe Arg Leu Arg Thr Asp Ile Lys Leu Tyr 180 185 190 Glu Cys Tyr 195 107 939 DNA Linum usitatissimum CDS (1)..(939) 107 atg ggg cgg tgc aga gtt ctg gtg gtg gga ggt acc ggg tac ata ggc 48 Met Gly Arg Cys Arg Val Leu Val Val Gly Gly Thr Gly Tyr Ile Gly 1 5 10 15 aag cgg atc gtc aag gct agc atc gaa cac ggc cac gac act tac gtc 96 Lys Arg Ile Val Lys Ala Ser Ile Glu His Gly His Asp Thr Tyr Val 20 25 30 ctc aag cga cct gag acg ggg ctc gat att gaa aaa ttc cag ctc ttg 144 Leu Lys Arg Pro Glu Thr Gly Leu Asp Ile Glu Lys Phe Gln Leu Leu 35 40 45 ttg tct ttc aag aaa cag ggc gcc cac ctc gtc gag gcc tcc ttc tct 192 Leu Ser Phe Lys Lys Gln Gly Ala His Leu Val Glu Ala Ser Phe Ser 50 55 60 gac cac gag agc ctt gtt cga gcg gtg aag cta gtc gat gtc gtg ata 240 Asp His Glu Ser Leu Val Arg Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 tgt acc gtc tcg ggg gct cat tca cgc agc ctc ctc ctc cag ctc aag 288 Cys Thr Val Ser Gly Ala His Ser Arg Ser Leu Leu Leu Gln Leu Lys 85 90 95 ttg gtc gag gcc atc aaa gag gcc gga aat gtt aag aga ttc att ccg 336 Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe Ile Pro 100 105 110 tct gag ttt ggg atg gac ccg gcg agg atg ggg gat gca ttg gag cca 384 Ser Glu Phe Gly Met Asp Pro Ala Arg Met Gly Asp Ala Leu Glu Pro 115 120 125 ggg agg gag acg ttc gat ctg aag atg gtg gtg agg aaa gcg atc gag 432 Gly Arg Glu Thr Phe Asp Leu Lys Met Val Val Arg Lys Ala Ile Glu 130 135 140 gac gcg aat atc ccc cac act tac atc tcg gcc aac tgc ttt gga ggt 480 Asp Ala Asn Ile Pro His Thr Tyr Ile Ser Ala Asn Cys Phe Gly Gly 145 150 155 160 tat ttc gtc ggc aat ctt tcg caa ctc gga cct cta acc cct cct tcc 528 Tyr Phe Val Gly Asn Leu Ser Gln Leu Gly Pro Leu Thr Pro Pro Ser 165 170 175 gat aag gtc acc atc tat gga gat ggc aac gtc aaa gtg gtg tac atg 576 Asp Lys Val Thr Ile Tyr Gly Asp Gly Asn Val Lys Val Val Tyr Met 180 185 190 gat gaa gat gat gtc gcc act tac acg atc atg acg ata gag gat gac 624 Asp Glu Asp Asp Val Ala Thr Tyr Thr Ile Met Thr Ile Glu Asp Asp 195 200 205 cgg aca ctt aac aag acg atg tac ttc cgg cca ccg gaa aat gtg att 672 Arg Thr Leu Asn Lys Thr Met Tyr Phe Arg Pro Pro Glu Asn Val Ile 210 215 220 act cat agg caa tta gtg gag act tgg gaa aag ctc tca ggc aac caa 720 Thr His Arg Gln Leu Val Glu Thr Trp Glu Lys Leu Ser Gly Asn Gln 225 230 235 240 ctt caa aag act gag ctt tct tca caa gac ttt ctt gca ctc atg gaa 768 Leu Gln Lys Thr Glu Leu Ser Ser Gln Asp Phe Leu Ala Leu Met Glu 245 250 255 ggg aag gac gta gcg gag cag atc gta ata ggg cac ctc tac cac att 816 Gly Lys Asp Val Ala Glu Gln Ile Val Ile Gly His Leu Tyr His Ile 260 265 270 tac tac gaa gga tgt ctc act aac ttt gac atc gat gct gac caa gat 864 Tyr Tyr Glu Gly Cys Leu Thr Asn Phe Asp Ile Asp Ala Asp Gln Asp 275 280 285 caa gta gaa gct tca agt tta tac cct gaa gtt gaa tac act cgt atg 912 Gln Val Glu Ala Ser Ser Leu Tyr Pro Glu Val Glu Tyr Thr Arg Met 290 295 300 aaa gat tat ctg atg atc tac ctt tga 939 Lys Asp Tyr Leu Met Ile Tyr Leu 305 310 108 312 PRT Linum usitatissimum 108 Met Gly Arg Cys Arg Val Leu Val Val Gly Gly Thr Gly Tyr Ile Gly 1 5 10 15 Lys Arg Ile Val Lys Ala Ser Ile Glu His Gly His Asp Thr Tyr Val 20 25 30 Leu Lys Arg Pro Glu Thr Gly Leu Asp Ile Glu Lys Phe Gln Leu Leu 35 40 45 Leu Ser Phe Lys Lys Gln Gly Ala His Leu Val Glu Ala Ser Phe Ser 50 55 60 Asp His Glu Ser Leu Val Arg Ala Val Lys Leu Val Asp Val Val Ile 65 70 75 80 Cys Thr Val Ser Gly Ala His Ser Arg Ser Leu Leu Leu Gln Leu Lys 85 90 95 Leu Val Glu Ala Ile Lys Glu Ala Gly Asn Val Lys Arg Phe Ile Pro 100 105 110 Ser Glu Phe Gly Met Asp Pro Ala Arg Met Gly Asp Ala Leu Glu Pro 115 120 125 Gly Arg Glu Thr Phe Asp Leu Lys Met Val Val Arg Lys Ala Ile Glu 130 135 140 Asp Ala Asn Ile Pro His Thr Tyr Ile Ser Ala Asn Cys Phe Gly Gly 145 150 155 160 Tyr Phe Val Gly Asn Leu Ser Gln Leu Gly Pro Leu Thr Pro Pro Ser 165 170 175 Asp Lys Val Thr Ile Tyr Gly Asp Gly Asn Val Lys Val Val Tyr Met 180 185 190 Asp Glu Asp Asp Val Ala Thr Tyr Thr Ile Met Thr Ile Glu Asp Asp 195 200 205 Arg Thr Leu Asn Lys Thr Met Tyr Phe Arg Pro Pro Glu Asn Val Ile 210 215 220 Thr His Arg Gln Leu Val Glu Thr Trp Glu Lys Leu Ser Gly Asn Gln 225 230 235 240 Leu Gln Lys Thr Glu Leu Ser Ser Gln Asp Phe Leu Ala Leu Met Glu 245 250 255 Gly Lys Asp Val Ala Glu Gln Ile Val Ile Gly His Leu Tyr His Ile 260 265 270 Tyr Tyr Glu Gly Cys Leu Thr Asn Phe Asp Ile Asp Ala Asp Gln Asp 275 280 285 Gln Val Glu Ala Ser Ser Leu Tyr Pro Glu Val Glu Tyr Thr Arg Met 290 295 300 Lys Asp Tyr Leu Met Ile Tyr Leu 305 310 109 24 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 109 ccntcngagt tcggnatgga tccn 24 110 21 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 110 ngtatatttn acttcngggt a 21 111 603 DNA Linum usitatissimum 111 tctgagtttg ggatggaccc ggcgaggatg ggggatgcat tggagccagg gagggagacg 60 ttcgatctga agatggtggt gaggaaagcg atcgaggacg cgaatatccc ccacacttac 120 atctcggcca actgctttgg aggttatttc gtcggcaatc tttcgcaact cggacctcta 180 acccctcctt ccgataaggt caccatctat ggagatggca acgtcaaagt ggtgtacatg 240 gatgaagatg atgtcgccac ttacacgatc atgacgatag aggatgaccg gacacttaac 300 aagacgatgt acttccggcc accggaaaat gtgattactc ataggcaatt agtggagact 360 tgggaaaagc tctcaggcaa ccaacttcaa aagactgagc tttcttcaca agactttctt 420 gcactcatgg aagggaagga cgtagcggag cagatcgtaa tagggcacct ctaccacatt 480 tactacgaag gatgtctcac taactttgac atcgatgctg accaagatca agtagaagct 540 tcaagtttat accctgaagt tgaatacact cgtatgaaag attatctgat gatctacctt 600 tga 603 112 681 DNA Linum usitatissimum 112 cattcacgca gcctcctcct ccagctcaag ttggtcgagg ccatcaaaga ggccggaaat 60 gttaagagat tcattccgtc tgagtttggg atggacccgg cgaggatggg ggatgcattg 120 gagccaggga gggagacgtt cgatctgaag atggtggtga ggaaagcgat cgaggacgcg 180 aatatccccc acacttacat ctcggccaac tgctttggag gttatttcgt cggcaatctt 240 tcgcaactcg gacctctaac ccctccttcc gataaggtca ccatctatgg agatggcaac 300 gtcaaagtgg tgtacatgga tgaagatgat gtcgccactt acacgatcat gacgatagag 360 gatgaccgga cacttaacaa gacgatgtac ttccggccac cggaaaatgt gattactcat 420 aggcaattag tggagacttg ggaaaagctc tcaggcaacc aacttcaaaa gactgagctt 480 tcttcacaag actttcttgc actcatggaa gggaaggacg tagcggagca gatcgtaata 540 gggcacctct accacattta ctacgaagga tgtctcacta actttgacat cgatgctgac 600 caagatcaag tagaagcttc aagtttatac cctgaagttg aatacactcg tatgaaagat 660 tatctgatga tctacctttg a 681 113 30 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 113 aacatttccg gcctctttga tggcctcgac 30 114 30 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 114 aaggtagatc atcagataat ctttcatacg 30 115 22 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 115 gtaatacgac tcactatagg gc 22 116 20 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 116 aattaaccct cactaaaggg 20 117 942 DNA Schisandra chinensis CDS (1)..(942) 117 atg acg aag ttg agt gag agc aag gtt ctg att gtg ggt ggc aca ggc 48 Met Thr Lys Leu Ser Glu Ser Lys Val Leu Ile Val Gly Gly Thr Gly 1 5 10 15 cac ata ggg agg agg ctg gtt aga gcc agt ctt gcc ctt aat cac cca 96 His Ile Gly Arg Arg Leu Val Arg Ala Ser Leu Ala Leu Asn His Pro 20 25 30 act tac gtc ctg ttt cga gag gag aat ttg aat gat atc gag aag atc 144 Thr Tyr Val Leu Phe Arg Glu Glu Asn Leu Asn Asp Ile Glu Lys Ile 35 40 45 gag ctt ctt ctg gat ttc aag caa aac ggt gct cgt ctt gtg atg gga 192 Glu Leu Leu Leu Asp Phe Lys Gln Asn Gly Ala Arg Leu Val Met Gly 50 55 60 tcg ttc gac aac cgg gag agc ctg ctg aat gca gtt aag cag gtg gac 240 Ser Phe Asp Asn Arg Glu Ser Leu Leu Asn Ala Val Lys Gln Val Asp 65 70 75 80 atc gtc ata tcc gcc ttg gct gca aac cat gtc cgc cat gag atc atc 288 Ile Val Ile Ser Ala Leu Ala Ala Asn His Val Arg His Glu Ile Ile 85 90 95 acg caa ttg aaa ctc ctg gat gtc atc ata gaa gcc ggt cat atc aag 336 Thr Gln Leu Lys Leu Leu Asp Val Ile Ile Glu Ala Gly His Ile Lys 100 105 110 agg ttc ata cct tca gag ttt gga atg gac cca gat ata atg gtt ggt 384 Arg Phe Ile Pro Ser Glu Phe Gly Met Asp Pro Asp Ile Met Val Gly 115 120 125 gct cta cct cca ggc aat aag aca ttt ata gat aaa agc aag gtc agg 432 Ala Leu Pro Pro Gly Asn Lys Thr Phe Ile Asp Lys Ser Lys Val Arg 130 135 140 cgt gca ata gaa gct gca gga gtt ccc cat acc tat gtc tct gca aat 480 Arg Ala Ile Glu Ala Ala Gly Val Pro His Thr Tyr Val Ser Ala Asn 145 150 155 160 tgc tac gct gca tat ttc gtc ggt ggc ctg ggc caa atc ggc cct ggt 528 Cys Tyr Ala Ala Tyr Phe Val Gly Gly Leu Gly Gln Ile Gly Pro Gly 165 170 175 tta atc cca tca cag gaa aaa gtt gcc ctc ttt gga gat gga gag gcc 576 Leu Ile Pro Ser Gln Glu Lys Val Ala Leu Phe Gly Asp Gly Glu Ala 180 185 190 aaa gtg ata tgg aat gat gag atg gac ata gca aca tat gtt ctt aaa 624 Lys Val Ile Trp Asn Asp Glu Met Asp Ile Ala Thr Tyr Val Leu Lys 195 200 205 gca gca gac gat cca cgg aca tta aac aag gca ata ttt atc aga cct 672 Ala Ala Asp Asp Pro Arg Thr Leu Asn Lys Ala Ile Phe Ile Arg Pro 210 215 220 cca gac aat ata ctt tct cag aga gag ctt gtg caa ata tgg gag aaa 720 Pro Asp Asn Ile Leu Ser Gln Arg Glu Leu Val Gln Ile Trp Glu Lys 225 230 235 240 cta att ggc cat gaa tta aag aaa aca aat att tca tct caa gag tgg 768 Leu Ile Gly His Glu Leu Lys Lys Thr Asn Ile Ser Ser Gln Glu Trp 245 250 255 ttg aaa tct atg gaa ggg atg ccc gag ggg ctg caa tta gca atg gca 816 Leu Lys Ser Met Glu Gly Met Pro Glu Gly Leu Gln Leu Ala Met Ala 260 265 270 cac aac ttt cat ata ttc tat gaa ggg tgt tta aca aat ttc cca gtt 864 His Asn Phe His Ile Phe Tyr Glu Gly Cys Leu Thr Asn Phe Pro Val 275 280 285 ggt gat gat caa gaa gct tcg aag ctt tac cca gaa gtc aga tac aca 912 Gly Asp Asp Gln Glu Ala Ser Lys Leu Tyr Pro Glu Val Arg Tyr Thr 290 295 300 tct atg gaa gaa tat ttg aag cga tat cta 942 Ser Met Glu Glu Tyr Leu Lys Arg Tyr Leu 305 310 118 314 PRT Schisandra chinensis 118 Met Thr Lys Leu Ser Glu Ser Lys Val Leu Ile Val Gly Gly Thr Gly 1 5 10 15 His Ile Gly Arg Arg Leu Val Arg Ala Ser Leu Ala Leu Asn His Pro 20 25 30 Thr Tyr Val Leu Phe Arg Glu Glu Asn Leu Asn Asp Ile Glu Lys Ile 35 40 45 Glu Leu Leu Leu Asp Phe Lys Gln Asn Gly Ala Arg Leu Val Met Gly 50 55 60 Ser Phe Asp Asn Arg Glu Ser Leu Leu Asn Ala Val Lys Gln Val Asp 65 70 75 80 Ile Val Ile Ser Ala Leu Ala Ala Asn His Val Arg His Glu Ile Ile 85 90 95 Thr Gln Leu Lys Leu Leu Asp Val Ile Ile Glu Ala Gly His Ile Lys 100 105 110 Arg Phe Ile Pro Ser Glu Phe Gly Met Asp Pro Asp Ile Met Val Gly 115 120 125 Ala Leu Pro Pro Gly Asn Lys Thr Phe Ile Asp Lys Ser Lys Val Arg 130 135 140 Arg Ala Ile Glu Ala Ala Gly Val Pro His Thr Tyr Val Ser Ala Asn 145 150 155 160 Cys Tyr Ala Ala Tyr Phe Val Gly Gly Leu Gly Gln Ile Gly Pro Gly 165 170 175 Leu Ile Pro Ser Gln Glu Lys Val Ala Leu Phe Gly Asp Gly Glu Ala 180 185 190 Lys Val Ile Trp Asn Asp Glu Met Asp Ile Ala Thr Tyr Val Leu Lys 195 200 205 Ala Ala Asp Asp Pro Arg Thr Leu Asn Lys Ala Ile Phe Ile Arg Pro 210 215 220 Pro Asp Asn Ile Leu Ser Gln Arg Glu Leu Val Gln Ile Trp Glu Lys 225 230 235 240 Leu Ile Gly His Glu Leu Lys Lys Thr Asn Ile Ser Ser Gln Glu Trp 245 250 255 Leu Lys Ser Met Glu Gly Met Pro Glu Gly Leu Gln Leu Ala Met Ala 260 265 270 His Asn Phe His Ile Phe Tyr Glu Gly Cys Leu Thr Asn Phe Pro Val 275 280 285 Gly Asp Asp Gln Glu Ala Ser Lys Leu Tyr Pro Glu Val Arg Tyr Thr 290 295 300 Ser Met Glu Glu Tyr Leu Lys Arg Tyr Leu 305 310 119 944 DNA Schisandra chinensis 119 atgacgaagc tgagtgagag caaggttctg attgtgggtg gcacaggcca catagggagg 60 aggctggtta gagccagtct tgcccttaat cacccaactt acgtcctgtt tcgagaggag 120 aatttgaatg atatcgagaa gatcgagctt cttctggatt tcaagcaaaa cggtgctcgt 180 cttgtgatgg gatcgttcga caaccgggag agcctgctga atgcagttaa gcaggtggac 240 atcgtcatat ccgccttggc tgcaaaccat gtccgccatg agatcatcac gcaactgaag 300 ctcctggatg tcatcataga agccggtcat atcaagaggt tcataccttc agagtttgga 360 atggacccag atataatgtt tggtgctcta cctccaggca ataagacatt tatagataaa 420 agcaaggtca ggcgtgcaat agaagctgca ggagttcccc atacctatgt ctctgcaaat 480 tgctacgctg catatttcgt cggtggcctg ggccaaatcg gccctggttt aatcccatca 540 caggaaaaag ttgccctctt tggagatgga gaggccaagt gatatggaat gatgagatgg 600 acatagcaac atatgttctt aaagcagcag acgatccacg gacattaaac aaggcaatat 660 ttatcagacc tccagacaat atactttctc agagagagct tgtgcaaata tgggagaaac 720 taattggcca tgaattaaag aaaacaaata tttcatctca agagtggttg aaatctatgg 780 aagggatgcc cgaggggctg caattagcaa tggcacacaa ctttcatata ttctatgaag 840 ggtgtttaac aaatttccca gttggtgatg atcaagaagc ttcgaagctt tacccagaag 900 tcagatacac atctatggaa gaatatttga agcgatatct atga 944 120 31 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 120 kgtgttygay gatccyatta cybtwgacaa c 31 121 30 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 121 tgrctamgta wactycctct acaaataaag 30 122 18 DNA Artificial Sequence Description of Artificial Sequence oligonucleotide 122 ctcgagtttt tttttttt 18 

The embodiments of the invention in which an exclusive property or privilege is claimed are defined as follows:
 1. An isolated nucleotide sequence encoding a dirigent protein, wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98, and 105, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98, and 105, under stringent wash conditions.
 2. An isolated nucleotide sequence of claim 1, wherein said isolated nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14, under stringent wash conditions.
 3. An isolated nucleotide sequence of claim 2 wherein said nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:13 and SEQ ID NO:15.
 4. An isolated nucleotide sequence of claim 2 consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14.
 5. An isolated nucleotide sequence of claim 1, wherein said isolated nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18, under stringent wash conditions.
 6. An isolated nucleotide sequence of claim 5 wherein said nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NO:17 and SEQ ID NO:19.
 7. An isolated nucleotide sequence of claim 5 consisting of a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18.
 8. An isolated nucleotide sequence of claim 1, wherein said isolated nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, and 34, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, and 34, under stringent wash conditions.
 9. An isolated nucleotide sequence of claim 8 wherein said nucleotide sequence encodes an amino acid sequence selected from the group consisting of SEQ ID NOS:21, 23, 29, 31, 33, 35, 78, 87 and
 94. 10. An isolated nucleotide sequence of claim 8 consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, 34, 77, 86 and
 93. 11. An isolated nucleotide sequence of claim 1, wherein said isolated nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO:98, or to the antisense complement of the nucleotide sequence of SEQ ID NO:98, under stringent wash conditions.
 12. An isolated nucleotide sequence of claim 11 wherein said nucleotide sequence encodes the amino acid sequence of SEQ ID NO:99.
 13. An isolated nucleotide sequence of claim 11 wherein said nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:98.
 14. An isolated nucleotide sequence of claim 1, wherein said isolated nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO:105, or to the antisense complement of the nucleotide sequence of SEQ ID NO:105, under stringent wash conditions.
 15. An isolated nucleotide sequence of claim 14 wherein said nucleotide sequence encodes the amino acid sequence of SEQ ID NO:106.
 16. An isolated nucleotide sequence of claim 14 wherein said nucleotide sequence consists of the nucleotide sequence of SEQ ID NO:105.
 17. A double stranded nucleic acid molecule encoding a dirigent protein, said double stranded nucleic acid molecule comprising a coding strand and a complementary non-coding strand, wherein the coding strand hybridizes to the oligonucleotide molecule of SEQ ID NO:120 in 50 mM KCL at 60° C., and the non-coding strand hybridizes to the oligonucleotide molecule of SEQ ID NO:121 in 50 mM KCL at 60° C.
 18. An isolated nucleotide sequence encoding a dirigent protein, wherein said isolated nucleotide sequence hybridizes to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98 and 105 under wash conditions of 1×SSC at 58° C.
 19. A replicable vector comprising a nucleotide sequence encoding a dirigent protein, wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98, and 105, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98, and 105, under stringent wash conditions.
 20. A replicable vector of claim 19 wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14, under stringent wash conditions.
 21. A replicable vector of claim 19 wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18, under stringent wash conditions.
 22. A replicable vector of claim 19 wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, and 34, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, and 34, under stringent wash conditions.
 23. A replicable vector comprising a nucleotide sequence encoding a dirigent protein comprising an amino acid sequence selected from the group consisting of SEQ ID NO:78, 87 and
 94. 24. A replicable vector of claim 23 wherein said nucleotide sequence encoding a dirigent protein is selected from the group consisting of SEQ ID NO:77, 86 and
 93. 25. A replicable vector of claim 19 wherein said nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO:98, or to the antisense complement of the nucleotide sequence of SEQ ID NO:98, under stringent wash conditions.
 26. A replicable vector of claim 19 wherein said nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO:105, or to the antisense complement of the nucleotide sequence of SEQ ID NO:105, under stringent wash conditions.
 27. A host cell comprising a replicable vector comprising a nucleotide sequence encoding a dirigent protein, wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98, and 105, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS: 12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98, and 105, under stringent wash conditions.
 28. A host cell of claim 27 wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NO:12 and SEQ ID NO:14, under stringent wash conditions.
 29. A host cell of claim 27 wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NO:16 and SEQ ID NO:18, under stringent wash conditions.
 30. A host cell of claim 27 wherein said nucleotide sequence hybridizes to a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, and 34, or to the antisense complement of a nucleotide sequence selected from the group consisting of SEQ ID NOS:20, 22, 28, 30, 32, and 34, under stringent wash conditions.
 31. A host cell comprising a replicable vector of claim
 23. 32. A host cell comprising a replicable vector of claim
 24. 33. A host cell of claim 27 wherein said nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO:98, or to the antisense complement of the nucleotide sequence of SEQ ID NO:98, under stringent wash conditions.
 34. A host cell of claim 27 wherein said nucleotide sequence hybridizes to the nucleotide sequence of SEQ ID NO:105, or to the antisense complement of the nucleotide sequence of SEQ ID NO:105, under stringent wash conditions.
 35. A method of expressing of dirigent protein in a suitable host cell comprising introducing into the host cell a replicable expression vector that comprises a nucleic acid sequence encoding a dirigent protein, wherein said nucleic acid sequence hybridizes to the antisense complement of a nucleic acid sequence selected from the group consisting of SEQ ID NOS:12, 14, 16, 18, 20, 22, 28, 30, 32, 34, 98 and 105, under stringent wash conditions, and expressing said encoded dirigent protein.
 36. A method of producing a dirigent protein comprising culturing a host cell of claim 27, under conditions suitable for expression of the dirigent protein by the host cell, and then recovering the dirigent protein.
 37. A method of producing a dirigent protein comprising culturing a host cell of claim 31 under conditions suitable for expression of the dirigent protein by the host cell, and then recovering the dirigent protein.
 38. A method of producing a dirigent protein comprising culturing a host cell of claim 32 under conditions suitable for expression of the dirigent protein by the host cell, and then recovering the dirigent protein. 